GFP和PI标记流式的问题

1、构建了载体，表达目的基因+GFP的融合蛋白（用的是PEGFP-N1），转染48小时后，处理细胞，百分之七十乙醇固定过夜，PI单染上流式测周期！由于，转染效率太低，通过了流式设门分别圈出了阴性和阳性细胞，再分别测其CELL周期变化！

有几个问题，想和大家一起讨论一下：

（1）在一些SCI文献中报道，通过MTT法，此目的基因表达，促进一些细胞（非我用的细胞系，我用的转染CELL，之前没有报道！）增殖，但是，从周期图来看，转进去的阳性CELL，反而停在G0/G1期了，与阴性CELL相比，增殖的S+G2/M期CELL明显减少！

这种矛盾怎么解释呢?

（2）难道是融合的GFP蛋白影响了目的蛋白的空间构象，从而影响了生物特性！

同一学校有人构建了同一目的基因的载体,表达非融合蛋白(带了HIS标签,非GFP蛋白)，稳定转染了和我同样的细胞系，流式测周期,结果是与对照相比，转染的℃ELL S+G2/M的细胞明显增多！和我的结果不一样！

GFP细胞上流式最好不要用乙醇固定，gfp很容易透出胞外的，导致阳性率细胞很少。

建议多聚甲醛固定，然后透化处理。

2、My protocol for GFP-PI Flow cytometry Analysis is just as the follows:

collect the media in one 15 ml tube

Treatment adherent cells with 0.4 ml 0.25% Trypsin-EDTA (6-well plate or T-25 cm2 flask) and add 0.8 ml complete media to stop it, collect them in the same 15 ml tube

centrifuge cells together with the media at 1000 rpm for 5 min at 4℃

Decant the supernatant rapidly

Wash 2× with ice cold PBS

Decant the supernatant rapidly, resuspend the pellet in the residual PBS and add 0.35 ml cold PBS, mix gently

Pipet it into 0.5 ml of 1% paraformaldehyde (ct = 0.5%) (1.5 ml Ep tube), vortex for 1-2 s

Incubate cell suspention for 20 min on ice in the dark

centrifuge cells at 1000 rpm for 5 min at 4℃

Decant the supernatant rapidly

Wash 2× with ice cold PBS (800 rpm for 5 min at 4℃)

Resuspend the pellet in the residual PBS, pipet it into 2 ml of 90% ethnol/10% PBS (-20℃ ) (15 ml tube) and vortex for 1-2s

Incubate cell suspention for 20 min on ice (probably not required)

centrifuge cells at 800 rpm for 5 min at 4℃

Decant the supernatant rapidly and washed 1× with 5 ml of PBT (PBS with 0.1% Triton X-100 and 0.5% BSA, store at 4℃)

Remove the supernatant with pipet carefully

Resuspend cells in 0.5 ml of PBT with 100 µg/ml RNase A and 50 µg/ml PI working solution except GFP-/PI-, GFP+/PI- control cells and incubate 1 h at 37℃ in the dark or overnight at 4℃

ethanol treatment, I think, can be neglected, because another people succeeded in this assay without
ethanol treatment. I will try it in my future experiment.

3、有两个问题

（1） ".......with 5 ml of PBT (PBS with 0.1% Triton X-100 and 0.5% BSA, store at 4℃)

" PBT在这里有何作用呢?不能用PBS洗涤吗?

I think Tritonx-100 and BSA in PBT is just for permeabilization and eliminating cell debris, respectively. I think if you do the step of ethanol permeabilization, then you ℃an use PBS to wash dire℃tly and PBT wash is not ne℃essary, anyway BSA is not so ℃heap.

（2）"with 100 µg/ml RNase A and 50 µg/ml PI working solution except GFP-/PI-, GFP+/PI- ℃ontrol cells and incubate 1 h at 37℃ in the dark or overnight at 4℃

" GFP-/PI-, GFP+/PI- control cells 的工作浓度又是多少呢?

sorry, this is my old protocol. at that time, I always set up GFP-/PI-, GFP+/PI- ℃ontrols. Actually, you need not set up these ℃ontrols. Only GFP-/PI+ ℃ontrol is demanded.