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Fixing cells for flow cytometry


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3 replies to this topic

#1 azazel

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Posted 02 August 2010 - 06:59 PM

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

#2 Clare

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Posted 03 August 2010 - 01:55 AM

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

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



#3 Piersgb

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Posted 03 August 2010 - 03:33 AM

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....CC/fixation.htm

#4 grayhair

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Posted 11 August 2010 - 08:26 AM

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.




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