flow cytometry protocol

[Jorg Kirberg]

In article <19971013205001.QAA09081 at ladder02.news.aol.com>, yfyang at aol.com
(YFYang) wrote:

> Does anyone have a protocol for detecting INTRACELLULAR antigen by using flow
>  cytometry?
> 
>  I tried using phosphate-citrate buffer to increase membrane permibility and
>  let antibody pass membrane, but I failed.
> 
Dear Ying,

from my few times experience, the following protocol worked fine. It's
compiled from several sources.

- start with a single cell suspension in FACS (PBS with 2 % FCS).
- fix cells with 1 % paraformaldehyde (I used a 4 % formaldehyde
  solution from our histology department and diluted it down) for
  5 - 20 min (determine empirically). Make shure you don't get clumps
  (e.g. add while slowly vortexing).
- wash cells with FACS
- wash once with FACS-SAP (FACS with 0.5 % saponine; some protocols
  ask only for 0.05 % saponine, others for 1 % (you have to try).
- add antibody in FACS-SAP at optimal concentration (to be determined
  before)
- wash with FACS-SAP. If you require a second step, start over with
  that one as in the step before.
- wash with FACS and analyse.

Good luck, jorg


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