Detection of IntracellularStaining using Flow cytometry - (Nov/12/2001 )
Would like to know the prefered method to fix cells for Immunofluorecence by Flow Cytometry.
Experiences with Paraformaldehyde Fixation is a lot of Smeary bits on the histogram. Is it possible to use ethanol or methanol to fix cells instead?
Another question, how do u permeabilise cells for intracellular staining without losing the intraceullar antigens.
thanks for your attention.
This is the method I use:
1. collect the cells, wash them and remove the supertanant
2. resuspend the cells in 2ml of cold intracellular staining buffer (4% phosphatebuffered paraformaldehyd/ 1% FCS) (worked fine!)
3. incubate for 10 minutes at 4°C
4. wash with PBS/ 10% FCS
5. add 2ml of permeabilisation buffer (permeabilisation concentrate: 100ml HEPES, 1M; 10g Saponin (Serva); filtrate sterile and store at 4°C) (permeabilisation buffer: PBS/ 1% FCS/ 1% perm concentrate; freshly mixed!)
6. incubate for 10 minutes at room temperature
The cells are ready for use!
CAUTION: Always use perm buffer for dilluting antibodies and washing the cells!!!
If you use FACS with introcellular staining, how do you exclude dead cells? So far, I suppose only Annexin V could do this job. But has anyone here ever tired?