This experiment is based on "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.
Operation method
Detection of Th1 and Th2 cells
Materials and Instruments
Intracellular factor antibody Move I. Test reagents For more product details, please visit Aladdin Scientific website.
PMA storage solution Enomycin storage solution Monensin RPMI-1640 Membrane breaker
1. PMA storage solution: PMA dissolved in DMSO, concentration of 0.1 mg/mI, -20 ℃ storage, working solution storage solution 1:100 diluted in RPMI-1640 or PBS (sterile, without sodium azide), at this time, 1 ug/ml, the working concentration of 25 ng/ml, 25 ug/ml of the original solution can be taken to dilute.
2. Enoximycin storage solution: Enomycin storage solution: Enomycin was dissolved in DMSO at a concentration of 1 mg/mI and stored at -20℃; working solution: storage solution was diluted 1:20 in RPMI-1640 or PBS (aseptic, without sodium azide), at this time, it was 50 ug/ml, and the working concentration was 1 ug/ml, and it was possible to dilute it by taking the original solution at 20 ug/ml.
3. Monensin: storage solution: Monensin was dissolved in methanol at a concentration of 50 mg/ml, and the working concentration was 1 ug/ml, and it was possible to dilute it by taking the original solution at 25 ug/ml. concentration of 50 mg/mI in methanol, in order to accelerate the dissolution, it can be placed in a water bath at 40~43 ℃. 4 ℃ only less than 4 months; working solution: storage solution 1:50 diluted in RPMI-1640 or PBS (sterile, without sodium azide), at this time, it is 1 mg/ml; the working concentration of 1.7 ug/ml (17 ul/ml).
4. RPMI-1640, RPMI-1640 containing 10% FCS (sterile, without sodium azide), can be diluted by taking 20 ug/ml stock solution. -1640 contains 10% FCS (fetal calf serum), 2 mmol/L glutamine 50 ug/ml penicillin, 50 ug/streptomycin and 100 ug/ml neomycin.
Antibodies: cell surface marker antibodies; CD3-PC5 (PerCP), CD8-FITC.
Intracellular factors: IL-4-PE, IFN-Y and others.
Membrane-breaking agents: Fix & Perm membrane-breaking agent from Caltag (USA); FACS membrane-breaking agent from BD (USA); IntraPrep membrane-breaking agent from Immunotech (France).
Steps
1. Heparin anticoagulation blood collection.
2. Take 2 test tubes as negative control tube (tube A) and assay tube (tube B), and add 100 uI blood and 100 uI PRM1-1640 to both tubes.
3. Add 3.4 ul 1 mg/ml Monensin working solution to tube A, and add 5 uI 1 ug/ml PMA working solution + 4 uI 50 ug/ml Ionomycin working solution to tube B. 50 ug/ml Ionomycin working solution + 3.4 ul 1 mg/ml Monensin working solution.
4. Incubate at 37℃, 5% CO2 incubator for 4~6 h.
5. Mix well, take out 100 uI, add appropriate amount of CD3-PC5 (PerCP), CD8-FITC, and incubate for a period of time (refer to the instruction manual of the antibody).
6. Fix and rupture the film with film-breaking agent.
7. Add appropriate amount of IL-4-PE, IFN-y PE, incubate for a period of time (refer to the instruction manual of the antibody).
8. Wash with PBS once, remove the supernatant; resuspend the cell precipitate with appropriate amount of PBS, and test on the machine.
Analysis of results
1. Select T lymphocytes by gating with CD3.
2. Analyze the lymphocytes identified in the first step by setting up a two-parameter histogram with CD8 and IFN-γ, respectively.
3. Derive the percentage of CD8/IFN-γ+ ( Th1 ) and CD8-/IL-4+ ( Th2 ) cells.