Fixing cells for flow cytometry - alternative to 1% paraformaldehyde (Aug/02/2010 )
is there an alternative to the last fixation step of staining cells using 1% paraformaldehyde? can i use formaldehyde instead? can anyone give me an alternative to 1% paraformaldehyde and its preparation formula as well? also, would it be okay to use RBC lysis instead of Facs Lysis to lyse the RBCs during the staining procedure? thanks
Hello 
You could try EtOH fixation? (depending on why you want to fix cells). I produced a monoclonal antibody to a transcription factor and found it only worked (well) when I EtOH fixed the cells (and not PFA). I fixed my cells in 70% EtOH (in PBS) ON at 4degC. Note: I stained cells the following day. Hope this helps!
Clare
azazel on Tue Aug 3 02:59:16 2010 said:
is there an alternative to the last fixation step of staining cells using 1% paraformaldehyde? can i use formaldehyde instead? can anyone give me an alternative to 1% paraformaldehyde and its preparation formula as well? also, would it be okay to use RBC lysis instead of Facs Lysis to lyse the RBCs during the staining procedure? thanks
What is the reason you want an alternative to PFA? My group tend to use 4% PFA in 1 x PBS for 10 minutes and then wash...
IHC world is usually quite good:
http://www.ihcworld.com/_protocols/general_ICC/fixation.htm
azazel on Tue Aug 3 02:59:16 2010 said:
is there an alternative to the last fixation step of staining cells using 1% paraformaldehyde? can i use formaldehyde instead? can anyone give me an alternative to 1% paraformaldehyde and its preparation formula as well? also, would it be okay to use RBC lysis instead of Facs Lysis to lyse the RBCs during the staining procedure? thanks
1% formaldehyde
30 mL methanol free formadehyde to 270 mL of 0.1% bovine serum albumin solution in phosphate buffered saline.
pH to 7.4+/-0.1
store at 4 degrees C in a dark bottle.