CFSE细胞增殖实验
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CFSE细胞增殖实验
Company Document number：WTUT-WT88Y-W8BBGB-BWYTT-19998
准备：
1. 20ml 预冷5%FBS(HI)-PBS
2. 50ml 预冷MACS buffer
3. 70um滤网
4. RBC lysis
5. LS column
6. 50ml 37度预热PBS
7. 50ml 37度预热complete culture medium(RPMI 1640 +10% Heat Inactivated
FBS+10 mM HEPES+1 mM penicilline streptomycin+ 50 mM 2-mercaptoethanol)
（一）CD3抗体包被
取96孔板每孔加入100ul 10ug/mlanti-CD3抗体（PBS） 密封4度过夜。
（二）淋巴细胞获取
1. 小鼠的脾脏结摘取于10ml 冷5%FBS-PBS中。
2. 于70um滤网研磨滤过，500g 4度 5min离心后弃上清。
3. RBC lysis裂红5 min，10ml 冷5% PBS终止。
4. 500g 4度 5min 离心后弃上清，10mlMACS buffer重悬，计数。留取105个细胞进行FACS。
（三） 磁珠分选nave CD8a+ T细胞
cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
cell pellet in 400 μL of MACS buffer per 108 total cells.
100 μL of Naive CD8a+ T Cell Biotin-Antibody Cocktail per 108 total cells.
well and incubate for 5 minutes in the refrigerator (28 °C).
5. Add 200 μL of MACS buffer per 108 total cells.
6. Add 200 μL of Anti-Biotin MicroBeads per 108 total cells. Add 100 μL of
CD44 MicroBeads per 108 total cells.
7. Mix well and incubate for an additional 10 minutes in the refrigerator
(28 °C).
8. Wash cells by adding 10ml of MACS buffer per 108 cells and centrifuge at
300×g for 10 minutes. Aspirate supernatant completely.
9. Resuspend up to 108 cells in 500 μL of MACS buffer.
10. Place LS column in the magnetic field of a suitable MACS Separator.
11. Prepare column by rinsing with 3 mL of MACS buffer.
12. Apply cell suspension onto the column. Collect flow-through containing
unlabeled cells, representing the enriched naive CD8a+ T cell fraction.
13. Wash column with 3 mL of MACS buffer. Collect unlabeled cells that pass
through, representing the enriched naive CD8a+ T cells, and combine with
the flow-through from step 3.
14. Determine cell number. 留取105个细胞进行FACS。
（四）CFSE预处理
1. Remove supernatant from the cell pellet.
2. Add CellTrace CFSE (1:1000 dilution in PBS) staining solution and gently
resuspend the cells to 1*106/ml.
3. Incubate at 37°C for 20 minutes, protected from light.
4. Add 4 volumes complete culture medium and mix.
5. Incubate at 37°C for 5 minutes.
6. Pellet the cells and remove the supernatant.
7. Resuspend the cell pellet in fresh, pre-warmed complete culture medium to
1*107/ml.
8.均分两份，其中一份加入2ug/ml anti-CD28，一份不加抗体。
9.包被板使用PBS洗涤一次，按105 -106 个细胞／孔梯度接种，培养3天后上机。
