1. Spleen Sample Preparation
1) Take a fresh mouse spleen sample and prepare it into a single-cell suspension. Centrifuge at 300 × g for 5 min and discard the supernatant.
2) Red blood cell lysis: Add 2 mL of 1× ACK Lysis Buffer [E-CK-A105] to resuspend the cells, and let stand at room temperature for 2 min. Note: Do not exceed 2 min of lysis to prevent excessive lysis; lysis at 4°C is recommended.
3) Immediately after lysis, add 12 mL of PBS buffer (self-prepared) or RPMI-1640 basal medium (self-prepared) to terminate the lysis.
4) Centrifuge at 300 × g for 5 min and discard the supernatant. Then add an appropriate amount of RPMI-1640 complete medium to resuspend the cells and perform cell counting. Adjust the cell density to 1 × 106 cells/mL.
2. Stimulation and Blocking (a stimulation and blocking kit (E-CK-A091) is required, self-prepared)
5) Reagent preparation (before preparation, centrifuge the dry powder at 8000-10000 × g for 1 min to collect the dry powder at the bottom of the tube before dissolving and preparing):
a) 500× Cell Stimulation MIX: Add 100 μL of Cell Stimulation MIX Solvent to each tube of Cell Stimulation MIX Powder (50 μg), and pipette thoroughly to mix to prepare 500× Cell Stimulation MIX.
b) 1000× Protein Transport Inhibitor MIX: Add 50 μL of 33% DMSO aqueous solution (self-prepared) to each tube of Protein Transport Inhibitor MIX Powder (200 μg), and pipette thoroughly to mix to prepare 1000× Protein Transport Inhibitor MIX.
6) Referring to the table below, add 500× Cell Stimulation MIX and 1000× Protein Transport Inhibitor MIX, and incubate the cells in a 37°C, 5% CO2 incubator for 5-6 h, mixing once every 1-2 h during the incubation.
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Experimental Grouping |
Experimental Content |
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Negative control group |
No treatment |
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Experimental group |
For every 1 mL of cell suspension, add 2 μL of 500× Cell Stimulation MIX and 1 μL of 1000× Protein Transport Inhibitor MIX |
|
Isotype control group |
7) Collect the cell suspension, centrifuge at 300 × g for 5 min, discard the supernatant, and collect the cell pellet.
8) Add 1 mL of Cell Staining Buffer [E-CK-A107], centrifuge at 300 × g for 5 min, and discard the supernatant.
9) Add an appropriate amount of 1× PBS to resuspend, perform cell counting, and then adjust the density to 1 × 106 cells/mL.
3. Set up sample tube grouping
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Sample Tube Setup |
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Viability Dye |
CD3 |
CD4 |
IL-17A |
iso-IL-17A |
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Blank |
To adjust instrument voltage |
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Viability dye single stain (if any) |
To adjust compensation |
✔ |
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CD3 single stain |
To adjust compensation |
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✔ |
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CD4 single stain |
To adjust compensation |
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✔ |
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IL-17A single stain |
To adjust compensation |
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✔ |
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Isotype control |
To eliminate background staining |
✔ |
✔ |
✔ |
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✔ |
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Fully stained tube |
Experimental results |
✔ |
✔ |
✔ |
✔ |
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4. (Optional) Amine-Based Viability Dye
10) According to the sample tube grouping, add 1 mL of cell suspension to each tube.
11) Add 1 μL of STYX™ Fixable Viability Dye working solution (Note: an appropriate amine-based viability dye should be selected according to the flow cytometry panel), gently pipette to mix, and incubate at room temperature or 4°C in the dark for 30 min.
12) Centrifuge at 300 × g for 5 min, discard the supernatant, add 1 mL of PBS to resuspend the cell pellet, centrifuge at 300 × g for 5 min, and discard the supernatant.
13) Resuspend the cells in 100 μL of Cell Staining Buffer [E-CK-A107] (at this point, the cell density is approximately 1 × 107 cells/mL).
5. Fc Receptor Blocking
14) Add 0.5-1 μg of purified anti-mouse CD16/32 monoclonal antibody [E-AB-F0997A], and incubate at room temperature for 10 min.
Note: No washing is required after incubation; surface antibodies can be added directly.
6. Surface Antibody Incubation
15) According to the experimental design, except for the blank control, add 5 μL of the corresponding surface staining antibodies (CD3, CD4) to each of the corresponding single-stain tubes and fully stained tubes, mix well, and incubate at 4°C in the dark for 30 min.
7. Fixation and Permeabilization (an intracellular cytokine fixation and permeabilization reagent (E-CK-A109) is required, self-prepared)
16) Reagent preparation: Permeabilization Buffer (5×) is a 5× concentrate. Before the experiment, dilute it with deionized water to 1× Permeabilization Working Solution, that is, take 1 mL of Permeabilization Buffer (5×) and add 4 mL of deionized water, mix well.
17) Add 2 mL of Cell Staining Buffer [E-CK-A107] to each tube, centrifuge at 300 × g for 5 min, and discard the supernatant.
18) Add 200 μL of Cell Staining Buffer [E-CK-A107] to resuspend the cells.
19) Add 200 μL of Fixation Buffer to each tube, mix gently, and incubate at room temperature in the dark for 30-60 min.
Note: If the entire detection process cannot be completed on the same day, after fixation, wash the cells twice with PBS, resuspend in PBS or basal medium, and store at 4°C, and perform the subsequent experiment on the next day.
20) Add 1 mL of 1× Permeabilization Working Solution, centrifuge at 600 × g for 5 min, and discard the supernatant.
8. Intracellular Antibody Incubation
21) Add 100 μL of 1× Permeabilization Working Solution to resuspend the cells.
22) Add 5 μL of intracellular antibody (IL-17A) to each of the corresponding single-stain tubes and fully stained tubes, and mix well.
23) Incubate at room temperature in the dark for 30 min.
24) Add 2 mL of Cell Staining Buffer [E-CK-A107] to each tube.
25) Centrifuge at 600 × g for 5 min, and discard the supernatant.
9. Acquisition on the Flow Cytometer
26) Add 200 μL of Cell Staining Buffer [E-CK-A107] to resuspend the cells.
27) Adjust the instrument parameters and acquire on the flow cytometer. Note: The flow cytometer voltage and threshold need to be set up before formal acquisition; after signal acquisition has started, the voltage and threshold must not be adjusted.
