流式分选求助！！！

羊芈芈

各位站友们，请问流式分选后的细胞活性怎么样？可以直接做后续的增殖实验（CFSE检测细胞增殖）还是说需要恢复活性（完全培养基孵育12h）后再做实验？本人做的是小鼠脾脏CD4+CD25-T细胞的流式分选，因为后续是要检测分选后的细胞的增殖，担心分选对细胞活性有影响，而且CFSE本身也对细胞有毒性，所以分选完的细胞到底该怎样处理呢?

niwanmao

一般建议培养一段时间。

BD有个Protocol供参考：
Sorted cell populations of CD45RA+ Tregs derived from 4 different donors were cultured for 13 to 14 days according to the procedure outlined by Basu et al.
Briefly, cells were cultured at a concentration of 2 x 105 cells/mL in X-VIVO 15 medium + 10% AB serum and stimulated with CD3/CD28 beads (cell:bead ratio of 1:1) in the presence of 100 ng/mL of rapamycin. Recombinant human IL-2 (300 units/mL) was added on day 2 and was present for the remainder of the culture period. Rapamycin was withdrawn on day 7 and cells re-stimulated with CD3/CD28 beads on day 9. Cell concentration was maintained at 2 x 105  cells/mL throughout the 13 to 14-day culture.
出处：https://www.bdbiosciences.com/do ... mp;_Measurement.pdf

羊芈芈

老师，您分享的这个Protocol我看了，做的是体外长期培养，长达2周，而且也没有提到分选后的细胞是直接用于后续实验呢。我昨天做了一次分选，把分选后的细胞培养基中过夜培养等其恢复活性后再进行后续实验。目前检测时间还没有到，但有种不好的预感