小中大
谢谢你们的答复！然而，在20天前，我们复苏同一批细胞，没有出现问题呀！（但是，这批细胞是储藏在-80度的低温冰箱里，已经一年了）大家说在24小时内，要是污染的话，培养基的颜色会不会仍旧是红色呢？另外，我将按照以下的步骤进行复苏，大家看看怎么样？多谢指导！
Thawing protocol of cryopreservated vero cell (this would be done as quickly as possible)
1) Pre-warm culture media in an incubator at 37℃ and place in the laminar flow hood. Obtain culture flasks, pipette, pipettor, tube and other necessary supplies and transfer to the hood.
2) Thaw vial quickly in a 37℃ water bath with constant, moderate agitation, until ice in the ampule is invisible. Note that immerse only the vial up to the cap to prevent leakage
3) Disinfect immediately the vial with 70% ethanol.
4) Work in a hood, open the vial and transfer the contents to a sterile 15ml tube.
5) Add 6ml of pre-warmed culture media containing serum and mix gently.
6) Centrifuge the suspended cells at 1500rpm for 10 min.
7) Decant the medium and resuspend gently the cell pellets in 6ml of culture medium and transfer into two 25 cm2 culture flasks and supplement the culture medium to 6ml of final volume for each flask.
8) Place them in an 37℃, 5% CO2 incubator.
9) After overnight incubation, the cells should be observed so that the viability may be evaluated. A trypan blue dye exclusion stain may be appropriate when precise viability assay is desired.
