发信人: John (Peter), 信区: Biology
标 题: Re: 有人做过immunofluorescence-based FACS吗?
发信站: BBS 未名空间站 (Fri Jul 28 22:53:20 2006)

1. If you have a Laser Scanning Cytometer around, use that for adherent
cells. That is a fantastic technology.

2.If you are trying to use FACS and do intracellular staining, use Saponin.
Triton is too harsh for FACs. Reason is simple, you need to push your cells
through the flow cell with high pressure, therefore the cells are easy
to be crashed. While microscopy studies don't have that problem. If your
antibody only worked under triton condition, try to run the samples on
a sorter so that you can control flow speed, decrease pressure.


【 在 bewithu (x-fire in the sea) 的大作中提到: 】
: 细胞是贴壁培养的,蛋白是胞内的,必须用indirect immunofluorescence,
: Last time, I tried as following,
: 1. Trypsinization
: 2. Make single-cell suspension
: 3. Add 4% PFA to the final conc. of 2%
: RT, 15 min
: 4. Centrifuge for 5 min at 2000 g
: Remove most of the sup, add the same volume of 0.2% Triton X-100
: RT, 15 min
: 5. Add more PBS, then centrifuge again
: ...................



--

※ 来源:·BBS 未名空间站 http://mitbbs.com·[FROM: 24.33.]