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流式细胞术可用来分析细胞的核酸含量，根据DNA含量，可区分静止G0期和增殖的G1期以及处于中间的S期。如果可同时检测细胞表型以及核酸含量 ，则可进一步细分出各类细胞的周期。本protocol介绍的就是在双激光（488nm、633或647nm）流式细胞仪上，同时检测2个表面抗原以及 DNA/RNA含量。DNA用皂素和7-AAD在低pH环境下染色，RNA则用PY（派若宁Y）。两种染料的浓度都很低，7-AAD与无荧光的废物线菌素D交换后，可明显减小对细胞表面染色的影响。
本protocol转自Cold Spring Harb Protoc 2007.
- Actinomycin D (AD) stock solution (1 mg/mL)
- 7-aminoactinomycin D (7-AAD) stock solution (1 mg/mL)
- Antibody staining solution
- Human cells of interest, fresh or cultured (or immortalized human cell lines)
- Monoclonal antibodies (mAb) directed against cell surface antigens of interest (biotinylated or labeled with an appropriate fluorochrome), and isotype-matched controls（本protocol中使用了一个生物素化单抗＋链霉素亲和AF488二抗，还有一个APC荧光的单抗进行表面双染。）
- 核酸染色液 (NASS，配方见下表) (pH 4.8)
Phosphate-citrate buffer tablets (Sigma-Aldrich)
Disodium EDTA (Sigma-Aldrich)
Bovine serum albumin, fraction V (Sigma-Aldrich)
为制备100 mL NASS (pH 4.8)，需在100ml蒸馏水中溶解两片磷酸盐 - 柠檬酸缓冲液片剂，然后再加入其它配方。溶液需保存在4°C.
Phosphate-buffered saline (PBS, 1X, without Ca++ and Mg++)
- Pyronin Y(G) (PY) stock solution (1 mg/mL)
- Streptavidin-Alexa Fluor 488
- Flow cytometer with 488-nm blue excitation and 633-nm or 647-nm red excitation, and the appropriate filter sets for collecting emissions from Alexa Fluor 488, PY, 7-AAD, and APC
- Incubator, preset to optimal temperature for cells of interest
1. Place 1 × 106 PBS-washed cells into a tube, add 100 μL of antibody staining solution, and mix well. In addition, prepare cells to be stained with isotype-matched control antibodies, as well as cells to be stained with a single color, to be used as controls during flow cytometry.
The preparation of samples stained with isotype-matched control antibodies is necessary for determination of background staining. The preparation of single-color control samples is necessary to determine the fluorescence overlap of APC and Alexa Fluor 488 into other detectors on the flow cytometer and to set accurate fluorescence compensation.
2. Label the cell-surface proteins of interest:
i. Add appropriate amounts of a biotinylated mAb and an APC-labeled mAb to the cell sample.
ii. Incubate the cells protected from light, using the optimal incubation temperature and time for the antibodies selected.
If the cell surface protein of interest is expressed at high levels, it may be possible to use a mAb that is directly conjugated to Alexa Fluor 488, omitting the need for indirect staining using a biotinylated antibody. If this is the case, omit Steps 3 and 4, and proceed directly to Step 5.
3. Wash the cells once by adding 2 mL of antibody staining solution and centrifuging at 250g for 5 min at 4°C. Discard the supernatant.
4. To the cell pellet, add 100 μL of antibody staining solution containing an appropriate amount of streptavidin-Alexa Fluor 488. Incubate for 20 min at 4°C.
5. Wash the cells once by adding 2 mL of antibody staining solution and centrifuging at 250g for 5 min at 4°C. Discard the supernatant.
6. Stain cells with 7-AAD:
i. Resuspend the cells from Step 5 in 0.5 mL of NASS containing 10 μg/mL of 7-AAD. Incubate for 20 min at 20°C-25°C, protected from light.
ii. Prepare a cell sample that is stained only with 7-AAD (without any cell surface staining).
This sample is necessary to determine the amount of fluorescence overlap of 7-AAD into other detectors on the flow cytometer and to set the appropriate fluorescence compensation.
7. Add 1 mL of 1X PBS to each sample. Collect the cells by centrifugation at 250g for 5 min. Discard the PBS.
8. Stain cells with PY:
i. Resuspend each cell pellet in 0.5 mL of NASS containing 10 μg/mL of AD. Place the mixture on ice for 5 min, protected from light.
ii. Add 5 μL of a 1:10 dilution of the PY stock solution (made with H2O), and vortex immediately. Keep the cells on ice, protected from light, for at least 10 min before sample acquisition on the flow cytometer.
It is possible to keep cells in the staining solution protected from light at 4°C for a maximum of 3 d before sample acquisition on the flow cytometer, without adverse effects.
iii. Prepare a sample of cells stained only with PY (without any cell-surface or 7-AAD staining).
This sample is necessary to determine the amount of fluorescence overlap of PY into other detectors on the flow cytometer and to set the appropriate fluorescence compensation.
9. Run samples on the flow cytometer using a low sample differential. Collect the fluorescence signals for cell-surface immunofluorescence with log amplification. Collect the DNA and RNA signals with linear amplification（注意采用低速获取，可得到更小的CV值，利于分析）
Application of the method for concurrent flow cytometric measurement of two cell-surface antigens and DNA-RNA content. Human peripheral blood mononuclear cells were cultured for 72 h, either in medium alone (A-D) or in medium containing 100 ng/mL of soluble CD28.2 (Biodesign) in the presence of 100 ng/mL of surface-bound OKT3 (Ortho Diagnostics) (E-H). (C and G) Analysis of CD5+CD8+ T cells; RNA/DNA dot-plots were gated on scatter as shown in row 1 (in A and E) and R1 (in B) or R2 (in F), respectively. (D and H) Analysis of CD5-CD8+ cells; RNA/DNA dot-plots were gated on scatter as shown in row 1 (in A and E) and on R3 (in B) or R4 (in F), respectively. After 3 d in culture without stimulation, CD8+ cells, irrespective of CD5 expression, did not leave G0. As expected, with CD3/CD28 activation, CD8+ cells that coexpressed CD5 (a human T lymphocyte antigen) proliferated, while CD5-CD8+ (NK cells) remained in G0. After 72 h of costimulation, only 9% of CD5+CD8+ T cells remained in G0; ~15% were in G1a, 21% were in G1b, and the rest were represented in the S + G2+M phases of the cell cycle.