1. Stimulation and Blocking (Self-provided Stimulation and Blocking Kit E-CK-A091 required)
1) Reagent preparation (before preparation, centrifuge the powder at 8000-10000 × g for 1 min to gather the 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), pipette thoroughly to mix, and prepare 500× Cell Stimulation MIX.
b) 1000× Protein Transport Inhibitor MIX: Add 50 μL of 33% DMSO aqueous solution (self-provided) to each tube of Protein Transport Inhibitor MIX Powder (200 μg), pipette thoroughly to mix, and prepare 1000× Protein Transport Inhibitor MIX.
2) Take 250 μL of fresh heparin-anticoagulated blood, add 250 μL of serum-free RPMI-1640 basal medium, and mix well.
3) Add 500× Cell Stimulation MIX and 1000× Protein Transport Inhibitor MIX according to the table below, and incubate the cells in a 37°C, 5% CO2 incubator for 5-6 h, mixing once every 1-2 h during the incubation.
|
Experimental Grouping |
Experimental Content |
|
Negative Control Group |
No treatment |
|
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 |
4) After the incubation, collect the cells, centrifuge at 300 × g for 5 min, discard the supernatant, add 1 mL of Cell Staining Buffer [E-CK-A107] or 1× PBS (self-provided), mix well, centrifuge at 300 × g for 5 min, and discard the supernatant.
2. Red Blood Cell Lysis
5) Add 100 μL of Cell Staining Buffer or 1× PBS to resuspend the cells, then add 2 mL of 1× ACK Lysis Buffer [E-CK-A105], and lyse on ice for 2-3 min. Centrifuge at 300 × g for 5 min, and discard the supernatant.
Note: Lysis is complete when the solution changes from turbid to clear. Please centrifuge promptly to avoid excessive lysis causing cell damage.
6) Add 1 mL of Cell Staining Buffer or 1× PBS to resuspend the cells, centrifuge at 300 × g for 5 min, and discard the supernatant.
7) Resuspend the cells with an appropriate amount of 1× PBS, then count the cells and adjust the cell density to approximately 1 × 106 cells/mL.
3. Setting Up Sample Tube Grouping
|
Sample Tube Setup |
Purpose |
Viability Dye |
CD3 |
CD4 |
IFN-γ |
iso-IFN-γ |
IL-4 |
iso-IL-4 |
|
Blank |
Adjust instrument voltage |
|
|
|
|
|
|
|
|
Viability Dye Single Stain (if available) |
Adjust compensation |
✔ |
|
|
|
|
|
|
|
CD3 Single Stain |
Adjust compensation |
|
✔ |
|
|
|
|
|
|
CD4 Single Stain |
Adjust compensation |
|
|
✔ |
|
|
|
|
|
IFN-γ Single Stain |
Adjust compensation |
|
|
|
✔ |
|
|
|
|
IFN-γ Isotype Control |
Eliminate background staining |
✔ |
✔ |
✔ |
|
✔ |
✔ |
|
|
IL-4 Single Stain |
Adjust compensation |
|
|
|
|
|
✔ |
|
|
IL-4 Isotype Control |
Eliminate background staining |
✔ |
✔ |
✔ |
✔ |
|
|
✔ |
|
Fully Stained Tube |
Experimental results |
✔ |
✔ |
✔ |
✔ |
|
✔ |
|
4. (Optional) Amine-based Viability Dye
8) According to the sample tube grouping, add 1 mL of cell suspension to each tube.
9) Add 1 μL of STYX™ Fixable Viability Dye working solution (Note: Select an appropriate amine-based viability dye based on the flow cytometry panel), pipette gently to mix, and incubate at room temperature or 4°C in the dark for 30 min.
10) 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.
11) Resuspend the cells in 100 μL of Cell Staining Buffer [E-CK-A107] (the cell density at this point is approximately 1 × 107 cells/mL).
5. Fc Receptor Blocking
12) Add 5 μL of EasyStain™ Human Fc Receptor Blocking Solution [E-CK-A171] 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
13) According to the experimental design, except for the blank control, add 5 μL of each corresponding surface staining antibody (CD3, CD4) to 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 (Self-provided Intracellular Cytokine Fixation and Permeabilization Reagent E-CK-A109 required)
14) Reagent preparation: Permeabilization Buffer (5×) is a 5× concentrate. Before the experiment, dilute it with deionized water to 1× Permeabilization Working Solution, i.e., take 1 mL of Permeabilization Buffer (5×) and add 4 mL of deionized water, mix well.
15) Add 2 mL of Cell Staining Buffer [E-CK-A107] to each tube, centrifuge at 300 × g for 5 min, and discard the supernatant.
16) Add 200 μL of Cell Staining Buffer [E-CK-A107] to resuspend the cells.
17) 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 for subsequent experiments the next day.
18) Add 1 mL of 1× Permeabilization Working Solution, centrifuge at 600 × g for 5 min, and discard the supernatant.
8. Intracellular Antibody Incubation
19) Add 100 μL of 1× Permeabilization Working Solution to resuspend the cells.
20) Add 5 μL of each corresponding intracellular antibody (IFN-γ, IL-4) to the corresponding single-stain tubes and fully stained tubes, and mix well.
21) Incubate at room temperature in the dark for 30 min.
22) Add 2 mL of Cell Staining Buffer [E-CK-A107] to each tube.
23) Centrifuge at 600 × g for 5 min, and discard the supernatant.
9. Flow Cytometry Detection
24) Add 200 μL of Cell Staining Buffer [E-CK-A107] to resuspend the cells.
Adjust the instrument parameters and perform flow cytometry detection. Note: The voltage and threshold of the flow cytometer need to be adjusted before formal acquisition. Once signal acquisition begins, the voltage and threshold must not be adjusted.
