准备：
1.20ml预冷5%FBS(HI)-PBS
2.50ml 预冷MACS buffer
3.70um滤网
4.2.5ml 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 mMpenicilline streptomycin+ 50 mM 2-mercaptoethanol)
（一）CD3抗体包被
取96孔板每孔加入100ul 10ug/ml anti-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。

3.2.5ml RBC lysis裂红5 min，10ml 冷5% PBS终止。

4.500g 4度5min 离心后弃上清，10ml MACS buffer重悬，计数。

留取105
个细胞进行FACS。

（三）磁珠分选naïve CD8a+ T细胞
1.Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
2.Resuspend cell pellet in 400 μL of MACS buffer per 108 total cells.
3.Add 100 μL of Naive CD8a+ T Cell Biotin-Antibody Cocktail per 108 total cells.
4.Mix well and incubate for 5 minutes in the refrigerator (2−8 °C).
5.Add 200 μL of MACS buffer per 108 total cells.
6.Add 200 μL of Anti-Biotin MicroBeads per 108total cells. Add 100 μL of CD44 MicroBeads per 108 total cells.
7.Mix well and incubate for an additional 10 minutes in the refrigerator (2−8 °C).
8.Wash cells by adding 10ml of MACS bufferper 108 cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
9.Resuspend up to 108cells in 500 μL of MACS bu ffer.
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 ofMACS 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 mediumto 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。