Sophie Dupré-Crochet

Marie Erard

Oliver Nüe
email: oliver.nusse@u-psud.fr

Kinetic analysis of phagosomal ROS generation

Keywords: NADPH oxidase, Phagosome, Microbial killing, Yeast, ROS sensitive dyes, Covalent labelling, DCF, DCFH2-SE, Imaging, Flow cytometry

Phagosomal ROS generation is critical for our immune defense against microbial infections.

Quantitative assessment of phagosomal ROS production is required to understand the complex relationship between the phagocyte and the microbe, in particular for pathogens that resist phagosomal destruction. ROS detection is difficult due to the transient nature of the reactive species and their multiple interactions with the environment. Direct labelling of phagocytic prey with a ROS sensitive dye allows to target the dye into the phagosome and to follow the kinetics of phagosomal ROS production on a single phagosome base. Here we describe the basic labelling procedure, the quality assessment and the imaging technique to achieve this kinetic analysis.

Introduction

The primary role of the phagocyte NADPH oxidase, NOX2, is the production of superoxide anion, which is converted to other reactive oxygen species ( ROS), inside phagosomes to kill ingested microbes [START_REF] Dupre-Crochet | ROS production in phagocytes: why, when, and where?[END_REF][START_REF] Nauseef | Biological roles for the NOX family NADPH oxidases[END_REF]. To this end, the phagocyte assembles and activates the enzyme specifically at the phagosomal membrane immediately upon formation of the phagosome. The importance of its role for host defense in infection becomes evident when NOX2 is absent or non-functional as in Chronic Granulomatous Disease [START_REF] O'neill | Genetic disorders coupled to ROS deficiency[END_REF]. Some pathogens either resist to ROS in the phagosome or inhibit NOX2 activation [START_REF] Imlay | Cellular defenses against superoxide and hydrogen peroxide[END_REF][START_REF] Seider | Interaction of pathogenic yeasts with phagocytes: Survival, persistence and escape[END_REF]. To investigate the mechanism of phagosomal ROS production and the associated pathologies, quantification of ROS inside the phagosome is required. From the wide choice of ROS detection methods only few are compatible with localized, time-resolved detection. The phagosome is particularly challenging because of the harsh intraphagosomal conditions in terms of changing pH, ROS, proteases and salt concentrations [START_REF] Nault | Environmental effects on ROS-detection -learning from the phagosome[END_REF].

Covalent labelling of the phagocytic prey with a ROS sensitive dye assures that the dye is localized inside the phagosome. Dichlorodihydrofluoresceine (DCFH2) is a fluorescein derivative that becomes fluorescent upon oxidation. Dichlorofluorescein (DCF) is the main oxidation product [START_REF] Tlili | Kinetic analysis of phagosomal production of reactive oxygen species[END_REF][START_REF] Wardman | Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: Progress, pitfalls, and prospects[END_REF]. DCFH2 is not specific and should be considered as a general indicator of ROS (see Note 1 and 2).We describe a general method to attach DCFH2 to heat killed yeast as well as other particles containing free amine groups. The procedure involves preparation of the particles, the labelling reaction, and a second reaction to de-protect the dye.

Since the dye is sensitive to chemical-and photo-oxidation all steps need to be performed at low oxygen tension and as little light exposure as possible. The quality of the preparation is best evaluated by flow cytometry. DCFH2-labeled yeast can be opsonized and used for timeresolved fluorescence microscopy or flow cytometry [START_REF] Dewitt | Phagosomal oxidative activity during beta2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2+ signal: Ca2+ controls time not space[END_REF][START_REF] Tlili | Stable accumulation of p67(phox) at the phagosomal membrane and ROS production within the phagosome[END_REF][START_REF] Song | Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome[END_REF]. Double or triple labelling of the particles with several dyes allows the detection of multiple parameters with the same particle [START_REF] Bernardo | Initial Cytoplasmic and Phagosomal Consequences of Human Neutrophil Exposure to Staphylococcus epidermidis[END_REF][START_REF] Russell | The macrophage marches on its phagosome: dynamic assays of phagosome function[END_REF]. We describe here the case of a reference dye that is rather stable in the phagosomal environment, which allows the independent quantification of phagocytosis. We will also provide a basic protocol for live cell imaging of phagosomal ROS production. The imaging conditions strongly depend on the features of the microscope. A number of controls are required to find the appropriate settings.

Methods

Particle labelling with DCFH2

The following protocol as outlined in Fig 1, has been developed for heat-killed yeast (Saccharomyces cerevisiae). We have also used this protocol for zymosan and Candida glabrata. In the literature, a similar labelling has been used for living microorganisms and for inert polystyrene beads [START_REF] Vanderven | Intraphagosomal measurement of the magnitude and duration of the oxidative burst[END_REF][START_REF] Kamen | SHIP-A Increases Early Oxidative Burst and Regulates Phagosome Maturation in Macrophages[END_REF][START_REF] Bernardo | Initial Cytoplasmic and Phagosomal Consequences of Human Neutrophil Exposure to Staphylococcus epidermidis[END_REF]. (see Note 5) 1. Yeasts, S. cerevisiae, are grown in YPD-medium at 30°C at 220 rotations per minute until they reach an exponential growth rate (absorbance at 600 nm between 0.2 and 0.4). They are spun down at 1100 x g and washed twice with PBS. They are resuspended at approximately 10 9 cells/ml and heat killed by boiling at >95°C for 60 min. Clumps are removed by filtration through filter paper of coarse porosity. Heat-killed yeast are washed in H2O, adjusted to 10 9 particles/ml and stored at -20°C. This preparation is stable for several years. (see Note 6) 2. For labelling, wash yeasts twice in bicarbonate buffer, centrifuge at 1100 x g.

3. Degas 10 mL bicarbonate buffer with nitrogen gas in a closed 15 mL Erlenmeyer flask for 60 min. The flask is closed with a rubber gasket pierced with 2 syringe needles. One of the needles is connected to the gas outlet and reaches into the buffer solution, the other does not reach into the buffer and serves for the escape of the gas from the flask. 4. Wash yeast once with degassed bicarbonate buffer.

5. Resuspend yeast in degassed bicarbonate buffer at 10 9 /mL and transfer into black Eppendorf vial sealed with a rubber gasket.

6. Degas Eppendorf vial with syringe needles through gasket 7. Add DCFH2-SE to yeast (250 µL dye solution for 10 9 yeast) by injecting through the gasket with a small (1 mL) syringe (see Note 7).

8. Incubate for 1 h at room temperature with constant gentle agitation and protect from light at all times (see Note 8).

Prepare the solution of hydroxylamine 1.5 M during the incubation time

Prepare 25 mL at 210 mg/mL of hydroxylamine in distilled water and adjust pH to 8.5 with a 5 M NaOH solution while stirring the solution. The solution will be at 3 M hydroxylamine and shall be diluted 1:2 to obtain the final solution at 1.5 M (see Note 9). [START_REF] Tlili | Stable accumulation of p67(phox) at the phagosomal membrane and ROS production within the phagosome[END_REF]. Add hydroxylamine solution directly to the yeast suspension to obtain 100 mM final concentration. Incubate for 45 min at room temperature. The hydroxylamine solution deprotects the ester groups of DCFH2 (Fig. 2) and neutralizes dye molecules that are not bound to the particles.

11. The yeast need to be washed intensively to remove unbound dye. Centrifuge for 5 min at 1100 x g and wash 3 times with degassed PBS. Recover the first supernatant for later testing (see Note 10).

12. Incubate in degassed PBS overnight at room temperature with gentle agitation.

13. Add 3 more washes with PBS and recover the last supernatant for testing. The sample is then incubated for 1 h at 37°C, washed twice with PBS and then resuspended in a suitable buffer for phagocytosis (see 3.3). The label does not seem to affect the outcome in terms of phagocytosis. Opsonized yeast are stored at -80°C. Even at that temperature, the opsonization is not stable and opsonized particles should not be stored for more than one month.

Assessment of labelling quality

The quality of the labelling is best assessed by flow cytometry. All particles should be labeled and the fluorescence intensity of the population should be homogenous (Fig. 3). However, a factor of 10 in fluorescence intensity between the weakest and the strongest particles is common. Treatment of the particles with H2O2 (50 mM) and HRP (50 U/mL) for 24 h at 22°C shifts the whole population to higher fluorescence [START_REF] Tlili | Kinetic analysis of phagosomal production of reactive oxygen species[END_REF]. For freshly labeled yeast, DCF fluorescence shifts by a factor of ≈ 10 from untreated to H2O2 / HRP-treated particles. (see

Note 11).

After double labelling, the fluorescence for both dyes detected by flow cytometry should be presented on a 2-parameter dot-plot. The points should form a cloud around a diagonal from left bottom to upper right [START_REF] Nault | Environmental effects on ROS-detection -learning from the phagosome[END_REF] indicating that particles that are weakly labeled with one dye tend to be also weakly labeled with the other dye. Strongly labelled particles are highly fluorescent with both dyes.

Time-lapse imaging of phagocytosis of DCFH2-labeled particles

Opsonized labeled yeasts are readily internalized by primary phagocytic cells and by neutrophil-like or macrophage-like cell lines. We have mostly used neutrophil-like, differentiated PLB985 cells. The actual imaging conditions depend strongly on the microscope setup. We will first describe a standard protocol and then list appropriate control experiments. 7. As the phagocyte itself and the phagosome within the phagocyte move in every direction, readjustment of the z-focus is often required. Therefore, the images have to be monitored during acquisition and the microscope focus adjusted accordingly.

8. End the recording if i) the phagosome is lost from focus or from the field of view, ii) significant bleaching occurs, iii) photooxidation of DCFH2 renders all yeast particles fluorescent, even those that are not internalized.

Control steps for imaging protocol

A number of controls are required to optimize this protocol for each microscopy setup. The appropriate exposure time for each image depends on the intensity of the excitation light, the quality of the optical path and the sensitivity of the camera. DCF is sensitive to photobleaching. DCFH2 is also sensitive to photooxidation that renders a fluorescent product. 1. To optimize between these two opposing constraints, it is useful to start with DCFH2labeled yeast alone to see how much light they support before photooxidation occurs.

2.

Then, DCF-yeasts should be prepared by deliberate oxidation (50 mM H2O2 and 50 U/mL HRP for 24 h) [START_REF] Tlili | Kinetic analysis of phagosomal production of reactive oxygen species[END_REF]. These oxidized yeasts will indicate the exposure time to obtain a signal with a satisfactory signal to noise ratio under the microscope.

3.

Repeated exposure of these DCF-yeasts will also indicate whether photobleaching occurs under the chosen conditions. Photooxidation is not a linear phenomenon, for example, 10 x 300 ms exposure does not have the same effect as 5 x 600 ms exposure.

4.

Control experiments either with cells that do not have a functional NADPH oxidase (PLB gp91 phox knock-out cells) or in the presence of an inhibitor of the NADPH oxidase will confirm that any increase in DCF fluorescence during the course of phagocytosis is indeed due to phagosomal ROS-production.

5.

The rapid and strong ROS production by most phagocytes may oxidize all available dye molecules on the particle. Thus the increase in DCF fluorescence may end due to dye saturation instead of the end of ROS production by the phagocyte [START_REF] Tlili | Kinetic analysis of phagosomal production of reactive oxygen species[END_REF][START_REF] Nault | Environmental effects on ROS-detection -learning from the phagosome[END_REF]. (see Note 14) 4. Notes 1. DCFH2 in its various forms is widely used since the 1960s [START_REF] Chen | 2′,7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy[END_REF]. It is by no means a perfect dye. It has poor specificity, it is photoactivated and photobleached, and it has a moderate pH sensitivity. In the phagosome, DCFH2 most likely detects H2O2 and HOCl [START_REF] Tlili | Kinetic analysis of phagosomal production of reactive oxygen species[END_REF]. Today however, it is the only ROS sensitive dye commercially available as a succinimidyl ester. It would be extremely useful to have other dyes available for covalent labelling. Amplex Red would be a good candidate. Hydrocyanines are an interesting option because they exist in several colors and the oxidized variants are frequently used for protein labelling. Their succimidyl-esters are commercially available (Lumiprobe: https://www.lumiprobe.com; Biotium, https://biotium.com). It may be possible to label particles with cyanine dyes and then reduce them to obtain ROS sensitive particle [START_REF] Kundu | Hydrocyanines: A Class of Fluorescent Sensors That Can Image Reactive Oxygen Species in Cell Culture, Tissue, and In Vivo[END_REF]. We are not aware that this has been performed to date.

2.

Genetically encoded biosensors could be an alternative to chemical labelling [START_REF] Erard | Biosensors for spatiotemporal detection of reactive oxygen species in cells and tissues[END_REF][START_REF] Schwarzlander | Dissecting Redox Biology Using Fluorescent Protein Sensors[END_REF].

Biosensors based on fluorescent proteins such as Hyper or roGFP are frequently used in redox biology. For measuring ROS production in the phagosome, the sensor should be placed on or near the surface of the living microorganism. The synthesis of the bacterial or fungal cell wall is a complicated issue that is only partially understood. Targeting of a biosensor to the cell wall should be possible but has not been reported. The known biosensors are reversible and interact with the cellular antioxidant machinery. It is not clear whether they would be appropriate for ROS detection in the phagosome. The harsh conditions of the phagosome (pH, proteases, ROS, salt) may substantially alter the photophysical properties of these sensors.

3.

The package size of DCFH2-DA, SE (5 mg) is larger than required for most phagocytosis applications. Weighing the dye to prepare aliquots most likely causes some oxidation. Taking up the whole package into a solvent (DMSO), preparing aliquots and evaporating the solvent maybe an option. We have not assessed whether this is feasible without a major loss of dye or reactivity.

4.

Particles such as yeasts are moving up and down in the Z direction along with the deformations of the phagocytes. In a wide field microscope, there is no spatial selection along the Z-axis and the fluorescence is integrated over a depth of several microns. On the contrary, with a confocal microscope, the axial sectioning leads to the detection of a single fluorescent plane. In confocal microscopy, the phagosome constantly moves in and out of the observation plane and renders any quantification difficult. If the reader wants to use a confocal microscope, an option is to record a Z-stack composed of a few planes at each time-point and to calculate the mean gray value of the yeast fluorescence for each plane. The highest value, corresponding approximately to the equatorial plane, could be taken for further quantification.

Labelling of live cells is possible, but faces 2 major problems. The actual labelling reaction

in bicarbonate buffer at pH 8 -9 is not toxic to many bacteria, yeast or even Leishmania (unpublished observation). The solvent DMSO may be toxic, but its concentration in the reaction medium can be reduced by using highly concentrated reagent solutions. On the contrary, the de-protection reaction with hydroxylamine is toxic to most microorganisms. Some references in the literature do not mention the second reaction. Avoiding the hydroxylamine step may be necessary to keep the cells alive, but the amount of "active" dye on the organisms may be much smaller. The second problem concerns the cost of the experiment. DCFH2-DA, SE (D2935 Molecular Probes) is sold in packages of 5 mg, a quantity enough to label >10 9 cells. Preparing aliquots of the reactive dye is difficult as light and oxygen should be avoided as much as possible. For dead particles, it is best to use the entire package in one reaction and store the labeled particle. Living bacterial or fungal cells cannot be easily stored. They may either die or proliferate. We do not know, whether storage in 25% glycerol at -80°C is compatible with method. So the labelling reaction has to be performed immediately before the experiment, which costs an entire package of the reagent and several hours of work (see also Note 2). 6. Several techniques for "heat killing" of yeast exist. We have used the same technique for two types of yeast (S. cerevisiae and C. glabrata) (see 3.1.1) and observed similar labelling results. An advantage of this method is the large number of sites available for the labelling.

The presence of a large number of attached dye molecules available for oxidation increases the amount of ROS detected before the saturation of the dye occurs and thus increases the detection window accessible for monitoring the labeled prey. Other heat killing protocols, namely lower temperature such as 70°C for 10 min [START_REF] Seider | The Facultative Intracellular Pathogen Candida glabrata Subverts Macrophage Cytokine Production and Phagolysosome Maturation[END_REF], may change the number of available amines for this reaction. The killing method also will change the surface of the yeast, which may affect the phagocytic process.

7.

Labelling with multiple dyes is possible. In particular, it is possible to label the yeast with Alexa 405-SE (Invitrogen A30000). Its fluorescence may serve as a reference in a ratiometric approach [START_REF] Nault | Environmental effects on ROS-detection -learning from the phagosome[END_REF][START_REF] Russell | The macrophage marches on its phagosome: dynamic assays of phagosome function[END_REF]. The fluorescence of Alexa 405 is quite stable with respect to pH or oxidation even within the phagosome. A solution of Alexa 405-SE (10 mg/mL in DMSO) can be added to the yeast together with DCFH2-SE. Typically, 10 µL of Alexa 405-SE solution are added to 10 9 yeasts in 1 mL at the same time as DCFH2-SE addition. Other succinimidyl ester dyes react under the same experimental conditions, butwhether they are stable inside the phagosome needs to be tested for each dye.

8.

Protection of DCFH2 against oxygen and light is tedious, but greatly improves the outcome.

Any oxidation of the dye during the reactions, washing steps and storage will increase the background due to DCF being bound on the particle. (see paragraph on quality assessment).

9.

Hydroxylamine is toxic, wear gloves and eye protection and dispose unused solution as chemical waste. The solution needs to be freshly prepared, at best during the first reaction period. The pH adjustment of this solution takes time. Concentrated, 5 mM NaOH needs to be added dropwise under stirring while monitoring pH with an electrode. If the solution is not properly stirred, pH may overshoot above 8.5 and then needs to be lowered by adding HCl. 10. At the end of the labelling procedure, yeasts are rose colored and the supernatant is light yellow due to unbound, oxidized dye. By oxidizing the dye (10 mM H2O2 + 10 U/ml HRP for 1 h) and measuring its absorbance (A), the concentration (c) can be determined. C = A / ( x l). l is the optical path length of the measuring cuvette, usually 1 cm. The extinction coefficient () of unbound DCF at pH 7.4 and 488 nm is 94,000 M -1 cm -1 [START_REF] Tlili | Kinetic analysis of phagosomal production of reactive oxygen species[END_REF]. The dye should be in excess to insure optimal labelling, thus at least some of the dye should remain unbound. Furthermore, at the end of the washing steps, no unbound dye should be detectable to make sure that the remaining dye is firmly attached to the particles.

11.

With time, DCFH2 oxidizes even under ideal storage conditions. It is worthwhile to test the basal and H2O2 / HRP-induced fluorescence by flow cytometer when older particles are used 12. DCF-particles are strongly fluorescent in the middle of the visual spectrum. Co-detection with fluorescent proteins is difficult since the latter tend to be weaker. Furthermore, excitation and emission spectra of DCF as well as fluorescent proteins are overlapping. Visualizing proteins tagged with mCherry in the same cell as fully oxidized DCF-particles has been possible in some but not all cases. Co-detection of DCF with the calcium indicator fura-2 is possible due to their clearly distinct excitation wavelengths [START_REF] Dewitt | Phagosomal oxidative activity during beta2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2+ signal: Ca2+ controls time not space[END_REF]. The red fluorescent pH indicator pHrodo is suitable for co-detection with DCF, even on the same particle [START_REF] Bernardo | Initial Cytoplasmic and Phagosomal Consequences of Human Neutrophil Exposure to Staphylococcus epidermidis[END_REF].

13.

Steps 5 and 7 require training. An experienced researcher will recognize cells that are likely to take-up a particle and he/she will lose less cells due to bad focus adjustment.

14.

The amount of label on each particle depends on the number of accessible amine groups.

Therefore, the type and size of the particle and its preparation method have a strong influence on the maximal signal that can be obtained. Phagocytes are strong ROS producers and can rapidly saturate the entire label on the particles.

Figures legends

  mM sodium bicarbonate buffer, 0.84 g NaHCO3 dissolved in 100 ml of water and then adjusted at pH 8.3 to suspend yeast during the reaction 2. Clean and dry DMSO 3.-1.5 M d'hydroxylamine solution at pH 8.5 prepared the same day 4.-DCFH2-SE (2′,7′-dichlorodihydrofluorescein diacetate, succinimidyl ester, Molecular probes D-2935) 5 mg, re-suspended in 0.5 mL DMSO, sufficient for 2x10 9 yeast (check appropriate number of particles when used with other microorganisms or beads) (see Note 3) 5.-Alexa405-SE (Molecular probes A30000) 1 mg, re-suspended in 0,1 ml DMSO, divide in 5 aliquots, evaporate DMSO in speedvac, storage at -20°C for approximately 6 months, resuspend each aliquot in 20 µL DMSO just before use. 6.-1 mL syringes, 23G syringe needles 7. Inert gas (N2 or Argon) from bottle or wall outlet 8.-Dark room with dim red light if possible 9. Centrifuge for yeast (1100 x g) 10. Slowly rotating wheel or shaker for tubes during reaction 11. Human serum (H4522,Sigma Aldrich) 12. Chamber for live cell microscopy 13. Flow cytometer with 488 nm laser 14. Wide-field inverted fluorescence microscope with appropriate filters for GFP or FITC detection (e.g; excitation at 480/15 nm, 495-nm beam splitter, and emission at 525/50 nm) and a fast, sensitive camera driven by a software for time-lapse acquisition (see Note 4)

14 .

 14 Store aliquots in light-protected vials under inert gas if possible at -20°C. Practically, aliquots of 20 µL with 10 9 yeasts/mL in eppendorff tubes are prepared and then briefly flushed with N2 and then closed.[START_REF] Kamen | SHIP-A Increases Early Oxidative Burst and Regulates Phagosome Maturation in Macrophages[END_REF]. Preparation for phagocytosis requires opsonization (in most cases). The labeled yeast are diluted 10 times and then opsonized with human serum. An equal volume of yeast and serum should be mixed (for example 50 µL of yeast at 10 9 yeasts/mL and 50 µL of human serum).

1 . 6 .

 16 Use an inverted fluorescent microscope equipped with filters for GFP or FITC detection (e.g; excitation at 480/15 nm, 495-nm beam splitter, and emission at 525/50 nm) and a sensitive rapid camera for fluorescence monitoring. (see Note 12) 2. Freshly prepare phagocytic cells (10 6 cells/ml) in a physiological buffer (e.g. 10 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM KCl) containing divalent ions (1 mM Mg 2+ , 2 mM Ca 2+ ), 1 mM glucose and a low concentration of protein (e.g. 1% human serum). Keep cells on ice. 3. Put cells on 25 mm coverslips fixed in a heated chamber (30 -37°C, below 30°C no phagocytosis occurs) in an inverted microscope in 100 µL buffer and let them settle on the coverslip. Add 300 µl of buffer in order to avoid dehydration. 4. Add 10 µL DCFH2-labeled yeast (at 10 8 yeast/mL) to the coverslip and let them settle on the phagocytes. 5. Chose a field with phagocytes surrounded by the yeasts. (see Note 13) Start time-lapse acquisition using an established protocol. Typically take images with 10 ms exposure at an interval of 10 s for a period of 30 min.

9 .

 9 Analyze data by choosing an ROI for each phagosome, adjusting this ROI to the xy-movement throughout the recording and measure mean grey value of the ROI for each image. Automated analysis is possible with the Multi-Dimensional Motion Analysis module from MetaMorph software (Molecular Devices) or similar analysis modules.

  Figure 1: Workflow of the labelling procedure. Step by step representation of the labelling

Figure 2 :

 2 Figure 2: Formula of 2',7'-dichlorodihydrofluorescein diacetate, succinimidyl ester

Figure 3 :

 3 Figure 3: Analysis of labeled particles by flow cytometry. Comparison of fluorescence

Figure 4 :

 4 Figure 4: Images of DCF-labeled yeast in phagosomes. Superimposed images of transmitted

Acknowledgement

This work was supported by grants from FRM (Foundation for Medical Research, DCM20121225747). We wish to thank our past colleagues who have contributed to this method and Elodie Hudik for excellent technical assistance.


Ngoc-Thao Noet
email: ngoc-thao.noet@etud.univ-angers.fr

Serge Blondel
email: serge.blondel@univ-angers.fr

Cooperation and ethical choices through an experimental approach

Keywords: cooperation, ethical values, public good game, prisoners' dilemma. JEL: C72; D12

Using a contingent valuation approach over 114 subjects, we assess the degree of cooperation and explain the factors underlying ethical choices. Implementing the prisoners' dilemma and public good games reveals a correlation between the amount of the ethical premium and the degree of cooperation. We identify factors that increase this cooperation, such as the frequency of interactions with individuals. The more cooperative individuals are, the higher the ethical premium. In addition, individuals naturally seek to cooperate. The more the game is repeated, the higher the degree of cooperation. According to the items, the degree of contribution is lower when dilution of responsibility occurs.

Introduction

The covid-19 crisis is no exception. Indeed, it has once again brought up to therefore the serious ethical questions encompassing sustainable economy matters, as well as the importance of cooperation in this field. This debate is mainly focused on the preservation of the common good, and the question of individual versus collective interest arises.

First, the challenge of responses to the covid-19 crisis (e.g., herd immunity) has shown that success can only be achieved through the ability to provide a global response, and thereby appeal to collective responsibility and cooperation. Most of the time, individual decisions lead to a modern version of the tragedy of the commons [START_REF] Gross | Individual solutions to shared problems create a modern tragedy of the commons[END_REF], and cooperation appears to be essential to meet collective challenges [START_REF] Axelrod | The Evolution of Cooperation[END_REF][START_REF] Axelrod | The Further Evolution of Cooperation[END_REF][START_REF] Hardin | The Tragedy of the Commons[END_REF].

If the dominant theories dismissed the question of ethics, behavioral economists have sought to nuance this rationality. Our judgment can be biased [START_REF] Hansen | The Definition of Nudge and Libertarian Paternalism : Does the Hand Fit the Glove?[END_REF][START_REF] Mongin | Rethinking Nudges[END_REF][START_REF] Thaler | From Cashews to Nudges: The Evolution of Behavioral Economics[END_REF][START_REF] Thaler | Nudge: Improving decisions about health, wealth, and happiness[END_REF] by emotions or moral values. The decisionmaker does not always act in a maximizing way. Recent studies have shown that individuals are complex [START_REF] Gregory-Smith | Emotions and dissonance in 'ethical' consumption choices[END_REF] and can choose to behave ethically [START_REF] Andersch | Understanding ethical purchasing behavior: Validation of an enhanced stage model of ethical behavior[END_REF][START_REF] Govind | Not Walking the Walk: How Dual Attitudes Influence Behavioral Outcomes in Ethical Consumption[END_REF][START_REF] Kluver | Behavioral ethics for Homo economicus, Homo heuristicus, and Homo duplex[END_REF][START_REF] Shaw | Care and Commitment in Ethical Consumption : An Exploration of the 'Attitude-Behaviour Gap[END_REF]. [START_REF] Kluver | Behavioral ethics for Homo economicus, Homo heuristicus, and Homo duplex[END_REF] define behavioral ethics as individual behavior that is subject to or judged according to generally accepted moral norms of behavior, while [START_REF] Shaw | Care and Commitment in Ethical Consumption : An Exploration of the 'Attitude-Behaviour Gap[END_REF] refer to ethics as a manifestation of caring, responsibility and felt obligation. We will use both these definitions and then refer to ethical behavior as a responsible, benevolent attitude that is subject to, accepted, and judged by moral standards.

In the UK, Co-op's unique Ethical Consumption Report that tracked total ethical spending each year over the past two decades shows that ethical consumption of personal products (including sustainable clothing) has increased from £909k to £1898k, or +109% from 2010 to 20203 . Phenomena such as whether and why consumers are willing to pay a premium for ethical products [START_REF] De Pelsmacker | Do Consumers Care about Ethics? Willingness to Pay for Fair-Trade Coffee[END_REF][START_REF] Kim | Premiums Paid for What You Believe In : The Interactive Roles of Price Promotion and Cause Involvement on Consumer Response[END_REF][START_REF] Tsarenko | Environmentally conscious consumption: The role of retailers and peers as external influences[END_REF][START_REF] Tully | The Role of the Beneficiary in Willingness to Pay for Socially Responsible Products: A Meta-analysis[END_REF] are widely analyzed in the literature. Even if consumers' behavior does not match their positive attitude towards ethical products, consumers were willing to pay a premium. De [START_REF] De Pelsmacker | Do Consumers Care about Ethics? Willingness to Pay for Fair-Trade Coffee[END_REF] demonstrated that for fair trade coffee this average premium was around 10%, while [START_REF] Tully | The Role of the Beneficiary in Willingness to Pay for Socially Responsible Products: A Meta-analysis[END_REF], with a meta-analysis of 80 research papers, revealed that the mean percentage premium is 16.8%. [START_REF] Kim | Premiums Paid for What You Believe In : The Interactive Roles of Price Promotion and Cause Involvement on Consumer Response[END_REF] found that a price reduction leads consumers to have an ulterior motive and to distrust the brand. Nevertheless, despite supporting the values of ethical consumerism, most consumers rarely stand behind their beliefs at the point of purchase. The translation from intentions to behavior reflects "ethical consumption gap" [START_REF] Carrington | Why Ethical Consumers Don't Walk Their Talk : Towards a Framework for Understanding the Gap Between the Ethical Purchase Intentions and Actual Buying Behaviour of Ethically Minded Consumers[END_REF][START_REF] Carrington | Lost in translation: Exploring the ethical consumer intention-behavior gap[END_REF][START_REF] Kilian | The Phantom of the 'Responsible Consumer' : Unmasking the Intention-Action Gap with an Indirect Questioning Technique[END_REF][START_REF] Shaw | Care and Commitment in Ethical Consumption : An Exploration of the 'Attitude-Behaviour Gap[END_REF]. Several factors have been identified such as habits, the priority given to ethics or the lack of opportunities, purchasing power, or time. In other words, ethical consumption means taking part in a collective action.

If [START_REF] Bennett | Social consensus, moral intensity and willingness to pay to address a farm animal welfare issue[END_REF] show the relationship between social consensus, moral intensity and people's willingness to pay (WTP), however, to the best of our knowledge, no study has considered altogether the degree of ethics in stated preferences and the degree of cooperation in non-cooperative games. First, this study highlights the relationship between individuals' willingness to cooperate and their commitment to their ethical choices. Second, it answers the question of how the use of the prisoners' dilemma game (and its variants) is a good predictor of ethical choices. To address this issue, we ran a questionnaire based on four motivation criteria: the eco-labels, the non-use of child labor, national manufacturing (in France), and biodegradable products in the first part. These four criteria cover all the major ethical themes and are consistent with our definition, respond to the ethically minded consumer behavior (EMCB) scale developed by [START_REF] Sudbury-Riley | Ethically minded consumer behavior: Scale review, development, and validation[END_REF] 4 , and allow to determine the stated preferences. In the second part, the implementation of the prisoners' dilemma (PD) and the public good (PG) games will reveal the correlation between the amount of the ethical premium and the degree of cooperation.

The remainder of this paper is structured as follows. Section 2 specifies our theoretical framework, hypotheses, and games modeling. Section 3 describes our survey. Section 4 shows our results and section 5 concludes.

Theorical framework, hypotheses, and games modeling

Theoretical framework and hypotheses

The individual does not always maximize. There are cognitive biases, emotions, and moral values. Cooperation requires a social dimension. Therefore, social preference is a concept that becomes closely linked to the notion of ethics. This behavior is illustrated in a sacrifice of one's own wealth or utility, to increase the wealth or utility of another person. Individuals are naturally cooperative [START_REF] Dawes | Anomalies: Cooperation[END_REF]) even when reciprocity was not possible, such as in one-shot experiments: the rate of cooperation was around 50% in PD games.

In the same way, many studies [START_REF] De Pelsmacker | Do Consumers Care about Ethics? Willingness to Pay for Fair-Trade Coffee[END_REF][START_REF] Kim | Premiums Paid for What You Believe In : The Interactive Roles of Price Promotion and Cause Involvement on Consumer Response[END_REF][START_REF] Tsarenko | Environmentally conscious consumption: The role of retailers and peers as external influences[END_REF][START_REF] Tully | The Role of the Beneficiary in Willingness to Pay for Socially Responsible Products: A Meta-analysis[END_REF] have shown that being interested in consuming ethically increases consumers' WTP. Based on this premise, after identifying the stated preferences, we can hypothesize that the degree of cooperation is correlated with the WTP. Our hypothesis (H1) is: the more cooperative individuals are, the higher the ethical premium is.

However, when faced with a decision, social preference is not always natural. This incompatibility between individual and collective interests was demonstrated by [START_REF] Hardin | The Tragedy of the Commons[END_REF] in the tragedy of the commons, in the use and sharing of a common resource and reveals the problem of the free riding [START_REF] Olson | The Logic of Collective Action : Public Goods and the Theory of Groups, Second Printing with a New Preface and Appendix[END_REF].

When it comes to social dilemmas and motivations, and when we try to understand their characteristics, the classical paradigm used is the PD game or/and its variants.

The prisoners' dilemma and the public good games

Understanding the drivers of cooperation between unrelated individuals remains a challenge for all social sciences. The PD and the PG game are the two most widely used standard models of cooperation.

The PD highlights that an individual action that does not lead to an optimum for the group. Nash equilibrium is reached when neither player cooperates, while the social optimum is reached when both cooperate. Hence, the PD is an important point for introducing ethical concerns, particularly through the link between cooperation, selfishness, and ethics [START_REF] Arce | Economics, Ethics and the Dilemma in the Prisoner's Dilemmas[END_REF].

In the same way, the PG game is a general version of PD. Formally, our PG game proceeds in the following way. 4 players participate. Each has an endowment E that he can divide between a collective investment ( and private . Thus, there is a public good that is multiplied by 2, then divided into 4 equal shares, regardless of the amounts of t . Thus, we have the individual payoffs for each player i ( ):

(1)

And the overall payoff for all players:

(2) The social optimum is obtained when all the endowments are put into the PG, which maximize P (2), with each person earning 2E. Conversely, the search for individual gain (1) leads to contributing nothing to a PG, with each player collecting only E. As in the PD, Nash equilibrium occurs when the contributions are null, while the social optimum is reached if all individuals contribute their full endowment.

However, experiments in the PG game show that subjects bet between 40% and 60% of their initial endowment and have a natural propensity to cooperate [START_REF] Chaudhuri | Sustaining cooperation in laboratory public goods experiments : A selective survey of the literature[END_REF][START_REF] Ledyard | Public Goods: A Survey of Experimental Research[END_REF]. However, multi-player PG game, unlike two-player PD, introduce a problem of dilution of responsibilities [START_REF] Darley | Bystander intervention in emergencies : Diffusion of responsibility[END_REF]). To initiate beneficial cooperation, a player must anticipate that all other players will reciprocate and that he or she is not left alone to make the effort for all (free rider problem). In the absence of a coordinating framework, cooperative strategies seem to be harder to implement in PG game (i.e., group interactions) than in the PD (i.e., pairwise interactions). Our within-subject design provides us the ability to analyze individual-level decisions obtained in different experiments with the same sample [START_REF] Blanco | A within-subject analysis of otherregarding preferences[END_REF]. The design with both games will allow to measure the dilution of responsibility effect. Our hypothesis (H3) is that the cooperation rate is higher in the PD game than in the PG game. Moreover, we have 2 treatments that allow us to play the PD twice, to understand if the repetition increases cooperation (H2).

Table 1. Hypotheses and tests

Tested by

Stated preferences PD PG H1: The more altruistic individuals are, the higher the ethical premium.

X X X

H2: Reiteration increases cooperation X H3: The cooperation rate is higher in the PD than in the PG X X

Experiment design

Survey design and data collection

The study was conducted online, among students at the University of Angers, and on the LinkedIn social network, from October 10, 2022, to October 25, 2022. The characteristics are gender, age (18+), study category (bachelor's or master's degree), and concerning the company: sector, size, and status. While there is little significant generational difference in work ethic [START_REF] Zabel | Generational Differences in Work Ethic: Fact or Fiction[END_REF](Zabel et al., ), generation Z (1996(Zabel et al., -2010) ) is still widely seen as the next consumer powerhouse [START_REF] Le | Ethical Consumption in Vietnam : An Analysis of Generational Cohorts and Gender[END_REF]. A link was made available to each participant, who could answer the questionnaire and send it in an anonymous way. Regarding the answers, as envisaged in AsPredicted5 , we removed an outlier (buying a -t-shirt for €1,000€ is an inconsistent response). The survey thus had a total of 114 respondents. The questionnaire was built with the Google Forms application and requires only 10 to 15 minutes. It was composed of 17 questions and divided into 3 parts. Nowhere is mentioned the word «ethics». The participants are simply told that they are invited to participate in a study on clothing buying motivations.

The first part is formed of 6 general questions and deals with their socio-demographic and economic profile. The second part is composed of two sets of 8 questions and deals with the ethical choices, as well as the indications of preference. We use a single-bounded dichotomous choice format (yes or no), with amounts in open-ended responses, for the WTP questions [START_REF] Bennett | Social consensus, moral intensity and willingness to pay to address a farm animal welfare issue[END_REF]. The last part was modeled by the two games: the PD and the PG, thus making it possible to analyze and understand the preferences towards the collective, as well as the degrees of cooperation. Our purpose here is being to understand the degree of ethics in stated preferences vs. the degree of cooperation, and as stated preferences were put in a hypothetical situation, it was the intention in this study to keep this hypothetical situation, and not the participants' performance, our survey does not provide any incentive [START_REF] Gneezy | Pay Enough or Don't Pay at All*[END_REF][START_REF] Voslinsky | Incentives in experimental economics[END_REF].

Prisoners' Dilemma

The first game is the standard PD, played in one round (PD1 7 ). The second is the repeated PD, played in two rounds in a row (PD2R1 8 and PD2R2 9 ).

For both versions, each player has a starting sum of €5,000 and have the choice of giving this money to the other player or keeping it. If he/she decides to donate, the amount is multiplied by 2. Thus, if both donate, both win €10,000. If both keep it, each one will only win his €5,000. Finally, if one keeps and the other gives, the first one earns €15,000 and the other one earns €0. 10 For the standard version, the game stops after the first choice. For the repeated version (PD2R1 and PD2R2), the rule is as follows: as soon as one of the players decides to keep a turn, the game stops automatically. If both give, both win €10,000, and the game continues. If one keeps, and the other gives, the first one wins €15,000 and the other wins €0, but the game stops. Therefore, for the game to go to the second round, both players must give, so each anticipates the benevolent cooperation of his opponent. To simulate the reaction of player 2, « give» or «keep», we made the game stop in G1 (

), whatever the choice of the player in the first round. In G2 (

), the other player always gives. We thus have 3 cases where the game stops and only 1 case where the game continues, which reflects real situations if the players were playing simultaneously.

7 PD1: PD Game 1 Round 8 PD2R1: PD Game 2 Rounds 1 st one 9 PD2R2: PD Game 2 Rounds 2 nd one 10 Regarding the games, we had two treatments: one group plays with strangers, while the other group plays with people they know. We observe no effect of this difference.

Figure 2. Prisoners' dilemma game simultaneous version

Public good game

Each participant is not able to communicate with the 3 other players. The parameters are and Specifically, each player can put €0, €1,000, €2,000, €3,000, €4,000 or €5,000 to the PG.

Survey results

General results

For this second part of the questionnaire, the stated preferences [START_REF] Andersch | Understanding ethical purchasing behavior: Validation of an enhanced stage model of ethical behavior[END_REF][START_REF] Czajkowski | Addressing empirical challenges related to the incentive compatibility of stated preferences methods[END_REF][START_REF] Mahmoudi | The preferences of consumers for organic tea : Evidence from a stated choice experiment[END_REF][START_REF] Vossler | Understanding the consequences of consequentiality : Testing the validity of stated preferences in the field[END_REF], are provided for our contingent valuation approach, which is used to reveal preferences and the WTP [START_REF] Diamond | Contingent Valuation: Is Some Number Better than No Number[END_REF]. We observe that the WTP is higher for products with a socially responsible element that benefits humans (i.e., labor practices) than for those that benefit the environment. This evidence corroborates the study by [START_REF] Tully | The Role of the Beneficiary in Willingness to Pay for Socially Responsible Products: A Meta-analysis[END_REF]. It may be noted that the criterion "no child labor" stands out considerably. The ethical themes are classified. Individuals are ready to make a real commitment when a theme affects them immediately. Other player… Respondents who give keeps (N=59) gives (N=55) PD1 (1) 55,9 54,5 PD2R1 (2) 72,9 63,6 PD2R2 (3) X 82,9 (a)

Table 3. Overall result ethical choices

(1) PD One Round, (2) PD Two Round, first one, (3) PD Two Round, second one. (a) N=31

In a second version of the prisoners' dilemma where the player can play twice, more participants trust the other player, 68,4% of the players give, against 55,3% when they play only once (p-value = 0,00652). This confirms H2: iteration increases cooperation.

Table 5. PG game results

Contribution to the public good

Total (%) €0 13,2 €1,000 23,7 €2,000 30,7 €3,000 13,2 €4,000 3,5 €5,000 15,8

On average, participants put 43.5% of the initial amount (i.e., €2,175) to the public good, which corresponds to the results of the literature with real payments. 87% of participants contribute to the public good, which reveals a strong willingness to cooperate. If we relate the amounts given in the two games, the correlation coefficient is equal to 0,283. The 63 subjects who have given in the PD, have given significantly more (2571.4) than the 50 ones who have kept (1686.3) The figure 1 illustrates this significant difference Since participants have given 43.5% of the initial amount in the PG and 55.3% in the PD, H3 is confirmed , the cooperation rate is cooperation is in % much stronger in a design with pairwise interactions than in a design with multi-interactions. In binary choices, the same individuals favor cooperation, although it involves more risk of losing (figure 2).

Figure 2. Difference between contribution and gain

Individuals are cooperative. 22% lost all of their bets. Among the individuals who decided to keep in the PD, 35% give in the PG game.

Econometric analysis

We use the ordinary least square method. Each premium, a continuous variable is treated as a dependent variable. Since PD1 give and PD2R1give are very strongly correlated at 45.1%, we only consider PD2R1Give in Table 6. These results confirm H1. There is a very significant correlation between the amounts of the ethical premiums and the cooperation. Regardless of the premium, this is true for the contribution to the PG game. For the first gift in the repeated PD (PD2R1), this is true for 2 premiums and coefficient is positive for the two others: this indicates that a long-term commitment increases the WTP and confirms a social motivation.

However, the cooperation is not the only factor that can explain the ethical choice. The amount of the ethical premium is strongly correlated with working for public administration, for all the premiums: being in the public sector reveals a willingness for collective action.

There might have many explanations. First, the values of « national manufacturing » directly reflect employment, the safeguarding of jobs, or the defense of a fairer employability. This is a value that makes sense and that directly affects individuals. In the PG game where responsibility is shared and where fear of the free rider may appeared, the fact that these values impact directly the subjects may have an impact on choice [START_REF] Slovic | If I Look at the Mass I Will Never Act: Psychic NumbingPsychic Numbing and Genocide[END_REF]. The individuals adopt familiar judgments, with a marked preference for the known or, on the contrary, an aversion to ambiguity.

When the PD game is repeated, the eco-label and Made in France criteria emerge. Individuals cooperate and trust, especially when there is a possibility for further interactions and especially the danger visible (unemployment and standard of living), individuals will seek to be cooperative and therefore prefer to play the collective game to preserve the relationship in the group [START_REF] Axelrod | The Evolution of Cooperation[END_REF][START_REF] Axelrod | The Further Evolution of Cooperation[END_REF][START_REF] Zhang | Common fate motivates cooperation: The influence of risks on contributions to public goods[END_REF].

Conclusion

Experiments using non-cooperative games are by default paid. As mentioned earlier, our goal is to understand the degree of ethics in stated preferences versus the degree of cooperation non-cooperative games. We have contextualized the stated preferences in a hypothetical situation, and we have kept it for the non-cooperative games. Because the goal of our study is to keep the consistency of stated preferences in the first game and their behavior toward strategic games, and not the performance of participants per se, our survey provides no incentives for strategic games [START_REF] Voslinsky | Incentives in experimental economics[END_REF]. In addition, since the topic of the survey is ethics and cooperation, compensation could have the opposite effect [START_REF] Gneezy | Pay Enough or Don't Pay at All*[END_REF] Our results thus show that ethical choices can be explained by cooperative behavior and illustrate the interest of the behavioral approach with both the modeling of non-cooperative games and stated preferences.

The more cooperative individuals are, the higher the ethical premium. In addition, individuals naturally seek to cooperate. The more the game is repeated, the higher the degree of cooperation. According to the items, the degree of contribution is lower in the case of dilution of responsibility. Furthermore, the OLS model reveals that some ethical criteria related to WTP are directly correlated with cooperation for the public good and the repeated prisoner dilemma games.

Social environment can have a strong influence on the ethical values affecting employment. The incorporation of these values by the various economic actors can be considered as a priority. On the one hand, because they are more identifiable/representative, which makes it easier to make ethical choices. On the other hand, this high rate of acceptance also makes it easier to reach the social optimum. However, although individuals integrate the ethical values related to the environment, there is only a little correlation regarding their behavior towards the defense of the collective (for the item biodegradable product). Nevertheless, in the repeated PD game we found a strong significance with the WTP ethical premiums for all four items. This shows that individuals cooperate especially in pairwise relationships, and when there is no dilution of responsibilities. They know why they participate in the collective but above all with whom they participate and that their actions count [START_REF] Darley | Bystander intervention in emergencies : Diffusion of responsibility[END_REF]. The issue of heterogeneity of meaning (including ethical/moral values) of participation in collectives is an important factor in behavior. Research perspectives on these items are topics for further refinement. Modeling strategic games in a hypothetical context with meaningful actions makes it possible to make some individual motivations salient, and not others. In this search for the preservation of a public good, the more targeted results may allow for a more precise adjustment of the actions of decision-makers before the implementation of public policy.

Annex: Survey

This questionnaire was run in French. It is translated into English here for the annex.

Preferences and choice behaviors

As part of an academic research project, we invite you to participate in a on clothing buying motivations.

This questionnaire should only take you 15 minutes at most. It consists of three parts:

-Part 1: general questions -Part 2: questions about your purchase motivations.

-Part 3: questions involving financial amounts.

You are asked to answer each question as if you were in the situation described. The results of the experiment are and will remain anonymous and confidential. For best results, please answer the given questions as fairly and reliably as possible and take the time to think carefully about what is being asked of you.

Game 1

In this 4-player game, you will play with 3 other players that you do not know and with whom you will not be able to communicate. A benefactor gives each of the players €5,000, if they participate. He explains the game rules to you. « You will each put whatever amount you want into the public good. I will multiply the total amount by two, and then I will divide it equally among the four of you, no matter how much you put in. So, you will each receive a quarter of the public good, plus the amount you kept

23.

For Survey Circle users, feel free to copy and paste the code below for your credits: ZB1Q-TQW3-NYPX-X3JX

24.

If you would like to add comments, feel free to use the slot below.

26.

To receive the results, please enter your e-mail address. *

Figure 1 .

 1 Figure 1. Amounts given in the PG game.

  25.If you would like to receive your results, please let us know.20Only one answer possibleo Yes, I would like to receive the results o No, I do not wish to receive the results

Table 2 . General characteristics of the survey (N=114) a

 2

	Variables	%
	Gender: Men	32,5
	Generation group:
	Generation Z 6	74,6
	Generation Y	14,9
	Generation X	8,8
	Baby-boomers	1,8
	Education:
	Youth Training/BTEC	0,9
	A Level	27,2
	Bachelor's degree 2	9,6
	Bachelor's degree 3	8,8
	Master's degree 1	19,3
	Master's degree 2	29,8
	PhD	4,4
	Activity:
	employee	21,9
	student	72,8
	other	5,3
	Company type:
	private	61,4
	public administration	21,9
	cooperative	3,5
	other	13,2
	Company size:
	Micro business < 10 FTEs	24,6

a the number of the sample

T-shirt at 15€ WTP a premium from a t-shirt … Rate of answer "yes" in % Average of the premium (%) Answer "yes"

				All
	with an eco-label	50,9	52,3	26,6
	with no child labors	81,6	63,7	52,0
	made in France	49,1	53,1	26,1
	with natural and biodegradable products	51,8	48,2	25,0

Table 4 . General results in the PD (in %)

 4

		Player 1
	Give	Keep
	Player 2	Player 2
	Give	Keep	Give	Keep
	(200,200)	(0,300)	(300,0)	(100,100)
	The game continues.		The game stops.

Table 6 .

 6 Dependent variable: Premium Amount PG game and PD game -OLS

	Premium

***, ** and *: significance at 1%, 5% and 10% levelsunilateral test for Contribution PG and PD2R1Give standard errors in parentheses. For each variable, the value is 1 if it is the situation described in the table, and 0 otherwise.

The only difference is that T-shirt B has an eco-label, unlike product

  The final results can be sent to you upon request, by filling in your e-mail address at the end of the survey. For any additional information, you can contact me at the following e-mail address: ngoc-thao.noet@etud.univ-angers.fr A. T-shirt A costs 15€. Are you willing to pay more for Tshirt B? * In this 4-player game, you will play with 3 other players that you do not know and with whom you will not be able to communicate. A benefactor gives each of the players €5,000, if they participate. He explains the game rules to you. « You will each put whatever amount you want into the public good. I will multiply the total amount by two, and then I will divide it equally among the four of you, no matter how much you put in. So, you will each receive a quarter of the public good, plus the amount you kept. » In this 2-player game, you will play twice with the same player you don't know, and with whom you will not be able to communicate. A benefactor gives each of you €5,000 if you participate. He explains the game rules to you. Each one keeps or gives to the other the €5,000, knowing that the donated amounts are multiplied by 2. But as soon as one of the players decides to keep in the 1st round, the game stops automatically.If both players give, both win €10,000, and the game continues. If both keep, you each win €5,000, and the game ends. If one keeps, and the other gives, the first wins €15,000 and the other wins €0, and the game stops.

	What is your level of education? You can choose between two similar t-shirts C and D. The only difference is that T-shirt D Part 3: Questions involving financial amounts
	Several answers are possible. does not use child labor, unlike C. T-shirt C costs 15€. Are you willing to pay more for T-Consider each of the situations in isolation
	o General certificate of secondary education or GCSE shirt D? *
	o Youth Training / BTEC First Diploma Game 1
	o A Level Only one answer possible.
	18.	o Bachelor's degree 2 o Yes, go to question 10
	o Bachelor's degree 3 o No, go to question 11 What do you do in the first round? *
	o Master's degree 1 Only one answer possible 10.
	o Master's degree 2 If yes, how much are you willing to pay at most (in €)? * o You keep the €5,000
	o PhD Go to question Go to question 23
		o Other: o You give the 5 000€
	16. The
	5. You can choose between two similar t-shirts E and F. The only difference is that t-shirt F is How much do you put in the public good? *
	What is the type of company you work for? * Made-in-France, unlike E. T-shirt E costs 15 €. Are you willing to pay more for T-shirt F? * Only one answer possible
	For students, this is the last company you worked for. Only one answer possible. o €0
	Only one answer is possible. o Yes, go to question 12 o €1,000
		o Private company o No, go to question 13 o €2,000
		o Public administration (including Chambers of Commerce and Industry, Regional o €3,000
	12.	Council, etc...) o €4,000
	o Association If yes, how much are you willing to pay at most (in €)? * o €5,000
	o Cooperative Go to question 13 Thank you in advance for your participation o Other Game 2
	13. In this 2-player game, you will play once with another player you don't know, and with whom *Mandatory You can choose between two similar t-shirts G et H. The only difference is that T-shirt H you will not be able to communicate. A benefactor gives each of you €5,000, if you participate.
	6. uses organic and biodegradable products, unlike G. T-shirt G costs 15€. Are you willing to He explains the rules of the game to you. Part 1: General questions Approximately how many people work (or worked) in the company, all locations combined? * pay more for T-shirt H? * Each of you keeps or gives to the other the €5,000, knowing that the donated amounts are
	Only one answer possible. multiplied by 2. 1. Part 2: Questions about your purchase motivations o Yes, go to question 12 If both give, both win €10,000. You are: * You walk into a clothing store. o No, go to question 13 If both keep, both win €5,000. Several answers are possible. If one keeps and the other gives, the first wins €15,000 and the other wins €0. o A man 7. 14. o A woman 2. Please indicate your age * If yes, how much are you willing to pay at most (in €)? * Go to question 15 15. 17. What do you do? * Only one answer possible o You keep the €5,000 You can choose between two similar t-shirts A and B. Only one answer possible. To be able to divide you into four groups, please indicate if the last 2 digits of your phone o You give the €5,000 3. o Yes, go to question 8 number (the 9th and 10th digits) are between: * You are: * o Between 75 and 99, go to question 19 o Other, go to question 4 If yes, how much are you willing to pay at most (in €)? * o Between 50 and 74, go to question 16 o Employee 8. o Between 25 and 49, go to question 19 o Student o Between 00 and 24, go to question 16 Only one answer is possible. o No, go to question 9 Only one answer possible. Game 3
	9. 4. Part 3 -Financial amounts (a)

other player decides to keep the €5,000. The game ends.

	Go to question 23
	Part 3 -Financial amounts (b)
	Part 3: Questions involving financial amounts
	Consider each of the situations in isolation

other player has decided to give the €5,000. The game continues. 22

  In this 2-player game, you will play once with another player you don't know, and with whom you will not be able to communicate. A benefactor gives each of you €5,000, if you participate. He explains the rules of the game to you. Each of you keeps or gives to the other the €5,000, knowing that the donated amounts are multiplied by 2. If both give, both win €10,000. If both keep, both win €5,000.If one keeps and the other gives, the first wins €15,000 and the other wins €0. In this 2-player game, you will play twice with the same player you don't know, and with whom you will not be able to communicate. A benefactor gives each of you €5,000 if you participate. He explains the game rules to you. Each one keeps or gives to the other the €5,000, knowing that the donated amounts are multiplied by 2. But as soon as one of the players decides to keep in the 1st round, the game stops automatically. If both players give, both win €10,000, and the game continues. If both keep, you each win €5,000, and the game ends. If one keeps, and the other gives, the first wins €15,000 and the other wins €0, and the game stops.21. What do you do in the first round? * Only one answer possible o You keep the €5,000, go to question 23 o You give the €5,000 The . What do you do in the second round? * Only one answer possible o You keep the €5,000, go to question 23 o You give the €5,000 Thank

	20.
	What do you do? *
	Only one answer possible
	o You keep the €5,000
	o You give the €5,000
	Jeu 3
	. »
	19.
	How much do you put in the public good? *
	Only one answer possible
	o €0
	o €1,000
	o €2,000
	o €3,000
	o €4,000
	o €5,000
	Game 2

you for your participation.

https://www.ethicalconsumer.org/sites/default/files/inline-files/EC_Market_Report_2021.pdf

The Sudbury-Riley and Kohlbacher's scale is defined in

dimensions: the purchase of environment-friendly products (ECOBUY); the boycotting of environmentally harmful products (ECOBOYCOTT); the purchase of products having recycled content (RECYCLE); the purchase of product based on social issues (CSRBOYCOTT); and the WTP more for an ethical product (PAYMORE).

https://aspredicted.org/B9B_652

Each generation has its own behavior pattern, which can be used as a repository for matching behaviors of the same generation[START_REF] Boyd | Ethical Determinants for Generations X and Y[END_REF]. Generation Z corresponds to 12 to 26 years old, generation Z to 27 to 42 years old, generation X to 43 to 57 years old and baby-boomers to 58 to

years old.