Paraformaldehyde detaches my 3T3 culture - (Feb/09/2006 )

Hello.

I am attempting to establish an immunofluorescence system in our lab but I am having trouble fixing my cells. I have tried to fix cultured NIH 3T3 fibroblasts using 4% paraformaldehyde (PFA) in PBS.
When I add the PFA the cells immeadiately detach, however, the cells do not detach from the PBS wash prior to the PFA addition.

Is it possible that my PFA percentage is too high?

Thanks

Hi thad93,

We once had this problem with sensitive cells. Although we couldnt get all of the cells to remain attached like other well-adhered cell lines we improved attachment by adding the first lot of 4%paraformaldehyde to the cells media (prior to PBS washes to avoid aggitating them). After 5 mins we tipped off the media/paraformaldehyde then repeated the paraformaldehyde fixation step.

Thats sounds like a great idea, I'll try it next week and let you know how it goes.

Thank you.