I've been optimising a flow cytometry protocol to assess intracellular proteins in human sperm. Most published methods describe fixing with 4%PFA after washing and pelleting the cells. I am finding the that after 10-30mins exposure to 4%PFA, when centrifuged, the cells clump together into a film and do not resuspend well, leaving to no visible pellet from then on and dramatic cell loss when analysed.
Sperm cells are prone to sticking together but it seems a common step in published sperm flow/IHC data, so I'm hoping someone has had this issue and solved! Are there any tips/tricks anyone knows of?
Hi John V. Stokes
Thanks for your answer. After writing I tried fixing in 2%, and had more cells and the pellet visibly clumped less, but still a large cell loss.
What I meant is that I had tried 10, 20, and 30 minute fixing times, always at RT, not that I leave it for a random amount of time in that range. I don't see a difference between these times.
Yes, in my hunt for answers I did read a really informative description of the nomenclature (I'll link below for anyone else interested), but went with the common abbreviation as it is what is used by the literature and is recognisable in the hope it would attract answers!
I'll continuing testing lower formaldehyde concentrations, thanks for the tip!
Best wishes,
Cara
http://www.cyto.purdue.edu/cdroms/cyto3/1/topics/prep/0011.htm
Hi, I am going to make a suggestion that may or may not work but worth trying anyway. Dextran can reduce aggregation of cells (eg blood cells). You may want to add some dextran 40 to the suspending solution. I would be interested to know whether it works.
Good luck
Siva
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