Key words

1 Introduction

The random nature of T cell receptor (TCR) gene rearrangement allows the generation of T cells recognizing essentially all pathogens, but also poses the threat of autoreactivity. A major mechanism upholding self-tolerance is negative selection in the thymus, usually in the form of clonal deletion. Since its first description more than 30 years ago [1], negative selection has been extensively studied by a variety of in vivo and in vitro approaches. The current gold standard for assessment of negative selection is the cross of TCR transgenic mice with mice expressing that TCR's ligand in the thymus (e.g., OT-1 × RIPmOva or OT-2 × RIPmOva) [2,3,4]. These models are invaluable in testing the importance of individual molecules for the ultimate success of negative selection; however, obtaining kinetics information from them is not straightforward. As a typical endpoint assay, one mouse provides a single data point and obtaining data on multiple time points and different treatments require large number of experimental animals.

Complementing the in vivo methods, a number of in vitro models have been used to study negative selection [5, 6]. These methods are usually easier to set up and multiplex. However, the absence of three-dimensional environment of the thymus with all supporting cell types as well as of key features of thymocyte behavior, such as motility, typically limits the utility of these models to assessment of cell death or signaling. Moreover, the lack of physiological survival factors results in high background level of apoptosis that can obscure small differences in the extent of negative selection.

A third group of methods, bridging the advantages of in vivo and in vitro assays, are the in situ models that use tissue explants or organotypic culture. Just like in the in vivo methods, negative selection takes place in thymocytes’ native three-dimensional environment that allows normal behavior and motility and is similar to the in vitro methods they are amenable to high-throughput analysis. Fetal thymic organ cultures (FTOCs) and re-aggregate thymic organ cultures (RTOCs) have been used to assess the contribution of different molecules or cell types to negative selection [6, 7]. Recently, a new in situ method is gaining popularity—the thymic slice. Vibratome-cut slices have all the elements of adult thymus environment, including fully developed cortex and medulla and are superior to FTOCs that represent fetal environment with underdeveloped medulla or RTOCs that do not have separate medulla and cortex. Additional important advantage of the slices is that they can easily be populated with a labeled population of interest by simply overlaying the cells for a couple of hours that allows them to migrate inside the tissue and behave in a very similar way to cells that have developed within the organ [3, 8]. Traditionally, marked cells could only be introduced into the thymus through the creation of bone marrow chimeric mice that usually requires at least 6 weeks. Moreover, it is not difficult to generate ~20 slices from a single thymus, which is enough to set up an experiment with six treatments or time points in triplicates using only one mouse. Thymic slices can easily be imaged and time-lapse two-photon microscopy is commonly used to observe the behavior of thymocytes and their supporting cell in the thymus and particularly in the medulla, which is difficult to access through the capsule of the organ due to its deeper position [3]. Similar to FTOCs and RTOCs, the viability of the thymocytes in the slice cultures is difficult to maintain for longer than 5 days, limiting long-term experiments. Thymic slices have been used to study positive selection [8,9,10], negative selection [3, 11,12,13], signaling in thymocytes [8, 14], regulatory T cell generation [15], and thymocyte motility [4, 16,17,18]. Several methods describing the use of thymic slices for observing the behavior of cells in the thymus by time-lapse two-photon microscopy have recently been published [19,20,21]. This chapter provides a detailed protocol to measure the kinetics and magnitude of negative selection using thymic slices and the OT-1/Ova257–264 system. OT-1 transgenic thymocytes can be stimulated to undergo negative selection by the encounter with their cognate peptide–Ova257–264 presented by H2-Kb [22]. The presence of a polyconal thymocyte population coming from C57BL/6 controls for any factors not related to negative selection and allows comparison between different experiments. Here, we describe how to prepare the thymic slices; isolate and label thymocytes and introduce them into the slice; and analyze the results by flow cytometry. The assay can be used to measure how a mutation or a particular treatment of thymocytes or thymus stroma affects the extent or kinetics of negative selection in situ.

2 Materials

  1. 1.

    70% alcohol in water (v/v).

  2. 2.

    Styrofoam dissection board.

  3. 3.

    Erlenmeyer flasks.

  4. 4.

    500 mL beakers.

  5. 5.

    10 cm forceps.

  6. 6.

    11 cm Iris scissors.

  7. 7.

    One-sided razor blades.

  8. 8.

    Metal spatula.

  9. 9.

    Paper towels.

  10. 10.

    Kimwipes.

  11. 11.

    70 μm nylon mesh (e.g., Small Parts, part # B0043D1SZG).

  12. 12.

    3 mL syringes.

  13. 13.

    6 cm Petri dishes.

  14. 14.

    6-well plates.

  15. 15.

    1.5 mL microcentrifuge tubes.

  16. 16.

    15 mL conical tubes.

  17. 17.

    5 mL polystyrene round bottom tubes (Falcon, cat. #352052).

  18. 18.

    Phosphate-buffered saline (PBS).

  19. 19.

    Hanks buffered salt solution (HBSS).

  20. 20.

    Complete Dulbecco’s modified Eagle medium (cDMEM): high-glucose DMEM supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/L penicillin, 100 μg/L streptomycin sulfate and 0.05 mM 2-mercaptoethanol.

  21. 21.

    Low-melting point agarose (e.g., NuSieve GTG Agarose, Lonza).

  22. 22.

    Disposable 22 mm embedding molds, square top tapered to 12 mm bottom (Polysciences, cat #18986).

  23. 23.

    Tissue glue (e.g., Vetbond, 3M).

  24. 24.

    Cell Proliferation Dye eFluor 450, stock solution at 5 mM in DMSO (eBioscience, ThermoFisher).

  25. 25.

    Cell Proliferation Dye eFluor 670 stock, solution at 5 mM in DMSO (eBioscience, ThermoFisher).

  26. 26.

    Ovalbumin (Ova257–264) peptide (Anaspec), stock solution: 1 μM Ova257–264 in DMEM, stored at −80 °C.

  27. 27.

    FACS buffer: PBS supplemented with 0.5% bovine serum albumin (BSA, w/v), 1 mM EDTA, and 0.1% sodium azide (NaN3, w/v).

  28. 28.

    Propidium iodide (PI) stock solution at 1 mg/mL.

  29. 29.

    0.4 μm pore size PET Cell Culture Inserts for 6-well plates (e.g., Falcon cat. #353090).

  30. 30.

    C57BL/6 mice (Jackson Labs, stock #000664).

  31. 31.

    OT-1 mice (NARLabs, Taipei, stock #RMRC13211 or Jackson Labs, stock #003831).

  32. 32.

    Microwave.

  33. 33.

    37 °C and 55 °C water baths.

  34. 34.

    Hemocytometer.

  35. 35.

    Refrigerating centrifuge (e.g., Eppendorf 5810R).

  36. 36.

    Vibratome (e.g., Leica VT 1000S).

  37. 37.

    CO2 incubator.

  38. 38.

    Flow cytometer equipped with violet (405 nm), blue (488 nm), and red (633 nm) lasers (e.g., LSR-Fortessa, BD Biosciences).

  39. 39.

    FlowJo flow cytometry analyzing software (BD Biosciences).

  40. 40.

    Microsoft Excel.

3 Methods

Note: All procedures used in animal experiments described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University, Taipei.

3.1 Preparation of Low-Melting Point (LMP) Agarose

  1. 1.

    Dissolve 2 g low-melting point agarose in 50 mL HBSS in a 250 mL Erlenmeyer flask for 4% final concentration. Add the agarose to the buffer, otherwise clumping might occur.

  2. 2.

    Boil in a microwave oven to completely dissolve the agarose. Wear heat-protecting gloves and avoid boiling over. In practice, we heat the mixture until it starts boiling and swirl to mix. We repeat these two steps until no trace of the agarose could be observed and the liquid is homogeneous and transparent.

  3. 3.

    Cover the flask with aluminum foil and put in a 56 °C water bath to cool down. Just before the dissection of the mouse, move the flask with the agarose to a 37 °C water bath (see Note 1).

3.2 Dissection of Thymus for Slices Preparation

  1. 1.

    Euthanize a C57BL/6 mouse by an institutionally approved method. We use CO2 suffocation followed by neck dislocation.

  2. 2.

    Place and secure the mouse on a dissection board on its back and saturate the abdomen and chest with 70% alcohol.

  3. 3.

    Using forceps and sharp scissors open up the peritoneal cavity and cut vena cava (see Note 2).

  4. 4.

    Pierce the diaphragm with scissors and cut it from the rib cage. Piercing of the diaphragm leads to collapse of the lungs.

  5. 5.

    Cut the side of the rib cage all the way to the armpits. Lift the sternum and the ribs and secure them with pins above the head to expose the mediastinum.

  6. 6.

    Carefully remove the thymus without squeezing or pulling it and place it in PBS on ice. We usually pull the heart that is attached to the thymus to separate the thymus from surrounding tissues. Then using sharp scissors, we cut around the thymus (see Note 3).

  7. 7.

    Clean the thymus of connective tissue: Place the thymus on wet paper towels and using small forceps and sharp scissors cut out all connective tissue. Keep the thymus always wet by spraying it occasionally with PBS. A good test for the thoroughness of cleaning is dipping the thymus in buffer. If there is any connective tissue left, it is usually clearly visible inside the buffer (see Note 4).

  8. 8.

    Separate the two lobes of the thymus and remove the connective tissue between them. Once the lobes are cleaned and separated, place them in a 6 cm dish with 5 mL of PBS on ice.

3.3 Embedding the Thymus in LMP Agarose

  1. 1.

    Place a 22 mm square tissue embedding mold in a beaker filled with water and crushed ice.

  2. 2.

    Fill the mold with LMP agarose to the point where the side walls become straight. Work quickly as the agarose will start solidifying soon (see Note 5).

  3. 3.

    Gently pick one thymus lobe for the capsule from the side that will be its top. Quickly dry the thymus by touching it with a Kimwipe. Submerge the thymus lobe in the agarose. Push it with the forceps until it sinks below the surface; otherwise the surface tension might prevent it from sinking (see Note 6).

  4. 4.

    Wait until the agarose solidifies and becomes translucent—around 1–2 min (Fig. 1a).

Fig. 1
figure 1

Preparation of a mouse thymus lobe for cutting with Vibratome. (a) Top view of a thymus lobe embedded in agarose. (b) The agarose block taken out of the mold and ready for trimming. Note that there are 2–3 mm of agarose between the thymus and the edge of the block. (c) Trimming of the agarose block in preparation for cutting with the Vibratome

Full size image

3.4 Cutting Slices with Vibratome

  1. 1.

    Put the mold with the thymus on a solid flat surface and press the bottom down with your thumb for several seconds. That should be enough for the agarose block to separate from the mold (see Note 7).

  2. 2.

    Put the agarose block on an elevated platform (the lid of a pipette tip box works well) and trim into a rectangular prism with a one-sided razor blade. Leave at least 2–3 mm on each side of the thymus and ~5 mm on the bottom side (see Note 8) (Fig. 1b and c).

  3. 3.

    Dry the bottom side of the agarose block with a Kimwipe.

  4. 4.

    Place a drop of tissue glue or superglue on the stage of the Vibratome and mount the agarose block on the glue. Press gently to ensure good adhesion. Secure the Vibratome stage in the cutting chamber.

  5. 5.

    Lower the blade just above the agarose block. Adjust the starting and finishing positions of the blade. Retract the blade to the starting position (see Note 9).

  6. 6.

    Fill the Vibratome cutting chamber with ice-cold PBS and the area around it with crushed ice.

  7. 7.

    Start cutting slices from the agarose block (Fig. 2a). We typically use the following settings on Leica 1000S: speed, 482 (~0.2 mm/s); amplitude, 1 mm; angle, 5°; and frequency, 8 (~80 Hz). Most often we cut slices that are 400 μm thick. We prefer to do single-slice cuts rather than continuous cutting, so that we can inspect each slice and make adjustments if necessary.

  8. 8.

    Once a slice is cleanly cut, carefully transfer it to a 6 cm dish with cDMEM on ice using a bent spatula (see Note 10) (Fig. 2b).

  9. 9.

    Keep the slices on ice until ready to overlay the thymocytes.

Fig. 2
figure 2

Cutting slices with the Vibratome. (a) Cutting of a 400-μm-thick thymic slice. (b) Retrieval of a cut slice with a spatula

Full size image

3.5 Harvesting and Labeling of Thymocytes

  1. 1.

    Euthanize a C57BL/6 mouse and an OT-1 mouse by an institutionally approved method (see Note 11).

  2. 2.

    Harvest the thymuses and clean them well according to Subheading 3.2 of the protocol (see Note 12).

  3. 3.

    Create single cell thymocyte suspension by smashing the thymus of each mouse with the back side of a plunger of a 3 mL syringe in 5 mL of PBS in a 6 cm dish.

  4. 4.

    Filter through 70 μm filter in a 15 mL conical tube (see Note 13).

  5. 5.

    Dilute the cell suspension to 10 mL with PBS.

  6. 6.

    Count the cells with a hemocytometer.

  7. 7.

    Take 10 × 106 cells of each cell suspension in fresh 15 mL conical tubes (see Note 14).

  8. 8.

    Spin down at 450 × g, 4 °C for 5 min. Aspirate carefully the supernatant.

  9. 9.

    Resuspend the cells from OT-1 mouse in 1 mL of pre-warmed to 37 °C PBS containing 1 μM Cell Proliferation Dye eFluor 450 (see Note 15). Vortex. Incubate at 37 °C for 15 min. Add 10 mL cDMEM medium and spin down at 450 × g, 4 °C for 5 min. Aspirate carefully the supernatant.

  10. 10.

    Resuspend the cells from C57BL/6 mouse in 1 mL of pre-warmed to 37 °C PBS containing 5 μM Cell Proliferation Dye eFluor 670. Vortex. Incubate at 37 °C for 15 min. Add 10 mL cDMEM medium and spin down at 450 × g 4 °C for 5 min. Aspirate carefully the supernatant.

  11. 11.

    Resuspend each cell suspension in 200 μL cDMEM medium so that the concentration is 50 × 106 cells/mL.

  12. 12.

    Mix the cell suspensions together so that the final volume is 400 μL and the concentration of each cell type—25 × 106/mL. Vortex to ensure good mixing of the cells.

3.6 Overlaying Thymocytes on Slices

  1. 1.

    In a biosafety cabinet, put Cell Culture Inserts into the wells of a 6-well plate.

  2. 2.

    Add 1 mL of cDMEM medium to the bottom of each well. This volume is enough to reach the membrane of the insert and keep the slices moist.

  3. 3.

    Carefully add three slices into one Cell Culture Insert with a bent spatula (Fig. 3a). Dry the slices before putting them inside by touching their edges to a Kimwipe (see Note 16). Make sure the slices do not touch each other or the walls of the Cell Culture Inserts.

  4. 4.

    Vortex the labeled cell suspension and carefully add 10–20 μL on top of each slice (see Note 17) (Fig. 3b).

  5. 5.

    Once all the slices have been covered with cell suspension, the 6-well plate should be covered with its lid and carefully moved to a CO2 incubator.

  6. 6.

    After 2 h, take the 6-well plate out and gently wash the cell suspension off the top of the slice with 1 mL of cDMEM medium (see Note 18). Remove the medium from the Cell Culture Insert by aspiration with a pipette.

  7. 7.

    Add 1 mL of cDMEM medium containing 10 nM Ova257–264 peptide to induce negative selection or 10 nM control peptide (see Note 19). Return back to the CO2 incubator.

  8. 8.

    After 30 min remove the peptide containing suspension by aspiration with a pipette. Add 10 μL of cDMEM medium on top of each slice to prevent them from drying during the continued incubation. Incubate for the desired time (see Note 20).

Fig. 3
figure 3

Overlaying of labeled thymocytes on cut thymic slices. (a) Arrangement of slices in a Cell Culture Insert. Note that the slices do not touch each other or the walls of the Cell Culture Insert. (b) Thymic slices overlaid with 10–20 μL labeled cell suspension that is retained on top of them

Full size image

3.7 Slice Dissociation and Flow Cytometry Analysis

  1. 1.

    After the end of the incubation period, take out the 6-well plate and add 1 mL of FACS buffer to the respective Cell Culture Inserts to facilitate the collection of the slices.

  2. 2.

    Use a bent spatula to move each slice to a 6 cm dish with 5 mL of ice-cold FACS buffer inside (see Note 21).

  3. 3.

    Create single cell suspension from the slice with the back side of a plunger of a 3 mL syringe. Make sure there are no pieces of intact tissue remaining.

  4. 4.

    Filter the suspension into 5 mL FACS tubes using pre-cut autoclaved 70 μm filters or cell strainer caps (see Note 22).

  5. 5.

    Move 1 mL of cell suspension to fresh FACS tubes (see Note 23).

  6. 6.

    Spin down at 450 × g 4 °C for 5 min. Aspirate carefully the supernatant.

  7. 7.

    Resuspend in 300 μL FACS buffer containing PI at 1:1000 dilution.

  8. 8.

    Proceed with the flow cytometry analysis using standard techniques. Acquire at least 200,000 cells, preferably 500,000 cells. This basic procedure requires flow cytometer with violet (405 nm), blue (488 nm), and red (633 nm) lasers. No compensation is required.

3.8 Analyzing the Flow Cytometry Data and Calculating the Cell Loss

  1. 1.

    We use FlowJo software for our flow cytometry analysis, so this workflow follows FlowJo’s conventions, but it can easily be adapted to any other software.

  2. 2.

    First gate on the live cells in FSC-A vs. PI plots and then gate on single cells in FSC-A vs. FSC-W plots.

  3. 3.

    Gate on the C57BL/6 cells and OT-1 cells in BV421 vs. APC plots (Fig. 4a).

  4. 4.

    Obtain the number of cells in each gate. For multiple samples this is easily done by exporting the cell counts to a table.

  5. 5.

    Copy the numbers to Microsoft Excel and divide the number of OT-1 cells by the number of C57BL/6 cells for each sample. This is the raw ratio.

  6. 6.

    Normalize the ratio to your control (usually control peptide stimulation)—obtain the average of the raw ratios for the control and divide all raw ratios by this number (see Note 24).

  7. 7.

    Plot the ratios for different treatments or time points and evaluate by an appropriate statistical method (Fig. 4b).

Fig. 4
figure 4

Representative data from an in situ negative selection experiment. (a) Example plots of OT-1thymocytes expressing red fluorescent protein (RFP) overlaid together with eFluor450 labeled C57BL/6 thymocytes (WT) on a C57BL/6 thymic slice and treated with control peptide (Ctrl) or Ova257–264 (Ova) peptide for 30 min. The slices were incubated overnight in a CO2 incubator, mechanically dissociated and analyzed by flow cytometry. The numbers are the proportions of cells in each gate out of total live, single cells. (b) Statistical analysis of the normalized ratio of live OT-1/WT thymocytes recovered from the slices. Error bars are standard error of the mean. N = 3 for control peptide and N = 6 for Ova peptide (∗∗∗∗p < 0.0001, unpaired t test)

Full size image

4 Notes

  1. 1.

    The agarose should be ~37 °C when the thymus is added in it. Keeping low melting point agarose at 37 °C for prolonged time can lead to solidification. The agarose can be stored and reused within a week. In this case it should be boiled again. However, we avoid boiling it more than two times, because the loss of water leads to increased agarose concentration and viscosity and, ultimately, difficulty in embedding the thymus.

  2. 2.

    The opening of vena cava decreases the chance of heavy bleeding during the dissection of the thymus.

  3. 3.

    Physical damage to the thymus such as cuts or squeezing of the parenchyma decrease the overall health of the tissue and can cause problems with migration or excessive cell death later and should be avoided as much as possible.

  4. 4.

    Thorough cleaning is critical for successful cutting of the thymus. Any remaining connective tissue on top of the capsule can interfere with separation of the slices by the Vibratome blade.

  5. 5.

    We suggest that every lab establishes optimal conditions for embedding the thymus. In our lab, we wait for ~20 s after pouring the agarose and then submerge the thymus in it. If you dip the thymus too soon, it will sink to the bottom and there will not be enough agarose on that side. If you wait for too long, the agarose will solidify and the thymus will not sink at all.

  6. 6.

    We typically submerge the thymus along its longest axis, but other orientations are possible. The guiding principle is that there should be enough agarose on each side once the agarose block is trimmed for slicing.

  7. 7.

    The separation of the agarose block can be facilitated if the corners of the mold are cut with a blade.

  8. 8.

    The agarose block can be trimmed in different shapes, but we prefer the rectangular prism. Each researcher should carefully evaluate the position of the thymus in the block and decide in advance which side should be bottom and which side should be top. The bottom side will be parallel to the cutting plane and should be smoothly cut. It should also have large enough surface to hold the whole block when glued to the Vibratome stage. Too small bottom sides detach easily during cutting, especially if the agarose block is tall. Because the Vibratome blade cannot reach the bottom of the stage, leave at least 5 mm between the bottom side of the agarose block and the bottom of the thymus. Do not leave too much agarose around the thymus, because the slices will be very big, difficult to cut cleanly, and difficult to position in a tissue culture insert later.

  9. 9.

    It is much easier to determine the starting and finishing position of the blade before the cutting chamber is filled with PBS.

  10. 10.

    Common problems during slice cutting are as follows: (1) The whole agarose block detaches from the cutting stage. In this case, the cutting chamber needs to be emptied of PBS and dried. Then the agarose block can be re-glued to the stage. (2) The blade cannot cut through the thymus and the slice remains attached to the agarose block through a thin piece of connective tissue. This is usually caused by suboptimal cleaning of the thymus lobes from connective tissue. The slice can be separated manually with scissors from the block; however, this usually results in defects in the slice. That is why it is advisable to prepare at least two thymuses in agarose blocks and very carefully clean the thymus from connective tissue. (3) The thymus slice detaches from the agarose. This could result from moving the thymus lobe once the agarose around it has started to solidify.

  11. 11.

    If you are doing an experiment with few slices (e.g., <8), it might be possible to use one lobe of the C57BL/6 thymus for slices and the other lobe for single cell suspension. In this protocol, we use OT-1 mice, because these are one of the most common TCR transgenic mice that have extensively been used to study negative selection. However, the method can be used with any TCR transgenic mice with a known cognate peptide.

  12. 12.

    The cleaning of the thymus for single cell suspension does not have to be as rigorous as for slice preparation. Leaving some connective tissue is acceptable, because it will be filtered out in subsequent steps. However, it is still advisable to remove any blood from the capsule by rolling the thymus on wet paper towels.

  13. 13.

    The cheapest option is to use pre-cut autoclaved filter membranes (e.g., Small Parts, part #B0043D1SZG or similar from Amazon). Alternatives include filtering into 5 mL polystyrene round-bottom (FACS) tubes with filter caps (Falcon, cat #352235) or into 50 mL conical tubes using 70 μm cell strainer (e.g., Falcon cat. #352350).

  14. 14.

    That number of cells will be enough for at least 20 slices. If the experiment needs to be scaled up, increase the number of cells accordingly.

  15. 15.

    Cell Proliferation Dyes 450 and 670 bind to free amino groups, so the buffer should not contain protein or TRIS, hence the use of PBS. Each lab is advised to titrate both dyes to find the optimal concentration for their purposes.

  16. 16.

    Careful drying is critical for the success of overlaying cells on the slice. If the slice is not dry, the surface tension cannot be maintained on its top and the cell suspension will leak to the membrane leaving no cells on top of the slice. Alternatively, a hydrophobic barrier such as vacuum grease silicon (e.g., Beckman, cat#335148) or Teflon O-ring (The O-Ring Store) can be used to make sure that the cell suspension will stay in place. The vacuum grease silicon can be applied with a syringe and a plastic needle. The O-rings can be bought in different sizes and one that fits the particular slice (surrounds all of the thymus tissue, but does not go outside of the agarose) can be put on top.

  17. 17.

    If the slice is dried well, the cell suspension will hold as a small drop on top of the slice. Avoid adding too much of the cell suspension as this will make it difficult for the drop to stay on top.

  18. 18.

    Careful, but thorough, washing is critical for the success of the experiment. Too little washing and many cells will be stuck on the top of the slice where their apoptosis will proceed with different kinetics compared to the cells inside the slice. The cells on top will likely be much more numerous than the ones inside and will dilute the effect of peptide or any other treatment. If there is no cell loss after specific peptide addition, this is the most likely step that needs to be optimized. Too much washing and the top layers of the slice will be washed away leaving very few labeled cells inside. We have found out that drop-wise addition of medium just above the thymus tissue while holding the plate tilted at 45° works best. Two or three rounds of such washing are usually sufficient.

  19. 19.

    The minimum concentration of agonist peptide can be as little as 1 nM [11], but more consistent results are obtained with 10 nM.

  20. 20.

    Depending of the purpose of the experiment, the incubation time can be different. In general, activation of OT-1 thymocytes could be seen as soon as 30 min after the peptide addition, signs of apoptosis (e.g., caspase activation, phosphatidylserine exposure) can be seen within 2 h of peptide addition, and cell loss can be observed starting after 3 h [11]. Approximately 40–60% of all OT I cells will be lost within the first 10 h. If the experiment is done with OT-1 × RAG1−/− mice, the cell loss will be even greater [23].

  21. 21.

    Make sure to label all dishes and tubes in advance. The incubation is good time to prepare them. Always confirm that you are using the correct containers.

  22. 22.

    The suspension will inevitably contain pieces of agarose, but we have found that they are readily filtered out and, typically, do not affect the flow cytometry analysis.

  23. 23.

    In our experience, the average slice has around 5 × 106 cells. In this case 1 mL of cells suspension will have ~106 cells. If the slice is very small or unusually big, it might be worth counting the cells to make sure that at least 106 cells are transferred for flow cytometry analysis. The entire cell suspension can also be used for flow cytometry. However, if further staining with antibodies is required, we recommend counting the cells in all samples and using equal number of cells for the staining.

  24. 24.

    The normalization is done to facilitate comparison between different experiments. Although every effort is made to make sure that the two cell populations are at 1:1 ratio, often times this is not the case, which can lead to raw ratios varying considerably between experiments and obscuring the majority of the differences.