CFSE assay for human peripheral blood T cell expansion in vitro
CFSE assay for human peripheral blood T cell expansion in vitro
CFSE detection of human peripheral blood T cell expansion in vitro refers to the use of CFSE to fluorescently label live cells, which can be used for the detection of cell proliferation, cell cycle estimation and cell division, etc. Here we only introduce the CFSE detection of human peripheral blood CD4+ T, CD8+ T and γδ+ T cell expansion technology in vitro.
Principle
The basic principle of CFSE for the detection of human peripheral blood T cell expansion in vitro is to utilize the property that CFSE can bind intracellular proteins to fluorescently label living cells. During cell division and proliferation, the fluorescence intensity of CFSE decreases step by step with cell division, and the labeled fluorescence can be evenly distributed to the two progeny cells, so its fluorescence intensity is half of that of the progeny cells. Based on this characteristic, it can be used to detect cell proliferation, cell cycle estimation and cell division.
Operation method
CFSE assay for human peripheral blood T cell expansion in vitro
Principle
The basic principle of CFSE for the detection of human peripheral blood T cell expansion in vitro is to utilize the property that CFSE can bind intracellular proteins to fluorescently label living cells. During cell division and proliferation, the fluorescence intensity of CFSE decreases step by step with cell division, and the labeled fluorescence can be evenly distributed to the two progeny cells, so its fluorescence intensity is half of that of the progeny cells. Based on this characteristic, it can be used to detect cell proliferation, cell cycle estimation and cell division.
Materials and Instruments
Equipment: Move The basic procedure for CFSE assay of human peripheral blood T cell expansion in vitro can be divided into the following steps: Caveat 1. The DMSO used to dissolve CFSE should be fresh and free of water, otherwise it will lead to the early hydrolysis of the AM body of CFSE, emitting green fluorescence and affecting the experimental effect. 2. CFSE working solution should be prepared and used now, because CFSE will decompose when absorbing water, which will affect the staining effect. Common Problems In order to determine the optimal staining concentration, it is recommended to refer to the relevant papers or do a pre-test for the final concentration of CFSE, set the final concentration as 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 μM ...... and then test the staining effect on the machine, to make sure that all the cells can be stained with CFSE, and if the concentration is too high, the proliferation will not be obvious. result in less obvious grouping after proliferation. For more product details, please visit Aladdin Scientific website.
Flow cytometer
Reagents:
① Live cell dye CFSE
② PBS
② PBS ③ 1640 medium containing 10% calf serum
④ PHA 10 μg/ml, antibody CD 35 μg/ml, Mycobacterium tuberculosis low molecular polypeptide antigen (Mtb - Ag) 5 μg/ml
⑤ IL-2 (50 U/ml)
⑥ Primary antibody as well as fluorescein-labeled secondary antibody, isotype control antibody
Consumables:
Flow-through tubes, centrifuge, glass tubes, 24-well plate.
1. Isolation of human peripheral blood mononuclear cells (PBMC).
2. Prepare cell suspension and adjust the concentration of the cells to about 1 x l07 ml-1 with suitable medium such as PBS.
3. Take 1 ml of cell suspension into a test tube, add appropriate amount of CFSE working solution, and gently mix well.
4. Incubate the cell suspension in an incubator at 37℃ for 15-30 min. 5. After centrifugation and supernatant removal, add 2 ml of 10% calf serum 1640 solution, then centrifuge and supernatant removal. Incubate in an incubator at 37 ℃ for 15-30 min.
5. Centrifuge and remove the supernatant, add 2 ml of 1640 solution with 10% calf serum, centrifuge again and remove the supernatant, and repeat this operation once.
6. Add suitable medium to make cell suspension.
7. Spread the plate (24-well plate, 1 x 106 per well).
8. Stimulate. Add PHA 10 μg/ml (PHA stimulation group), or antibody CD 35 μg/ml (CD3 stimulation group), or Mycobacterium tuberculosis low molecular polypeptide antigen (Mtb-Ag) 5 μg/ml (Mtb-Ag stimulation group), respectively, and incubate at 37 ℃, and then add IL-2 (50 U/ml) every 3 d for cell expansion.
9. Collect the cells that have been stimulated with PHA or CD3 mAb. Cells were cultured for 3d and 5d in the Mtb-Ag stimulation group, and collected after 8d, and stained for cell surface molecules with anti-CD4-PE, anti-CD8-PE, and anti-TCR78-PE. Then the cells were detected by flow cytometry with CFSE as the first fluorescence (FL-1) and PE-labeled antibody as the second fluorescence (FL-2), and the data were obtained by Cell Quest software, and then the proliferation dynamics of different T cell subsets were analyzed.