Hi,
Im in urgent need of some guidance regaring flowcytomatry. Im using this method to quantity the amount of spermatozoa and somatic cells in my samples. I have stain with Hoechst 33258, and hoped that the size wolud be sufficient to destinguish somatic from the sperm cells, BUT - in the case of sperm cells clumbing together this resulting in 2n, 4n etc - And I could not separete sperm form somatic.
Dose anyone have any experiance and could help me along my way?
I have been thinking about Vimentin for the somatic cells, and acrosome ab for the spermcells. Any thought or ideas?
All the best
In contrast with somatic cells, sperms are smaller in size and thus have a lower side-scatter light on scatter plot.
Thank you Farjam Goudarzi for your answer and your nice plot picture! For sure, my SSC/FSC do not look anything like that.
I am having problems identifying the correct population. I thinking about using Vimentin (in combination with Hoechst) to help identify the somatic cells - my secondary problem is that i do not have 100% control on the types of somatic cells in the sperm pellet squeezed from cauda and vas, only a general idea.
Do you know how sperms with and without tail will behave in the plot? Im using Hoechst so I think they will be in the same place since it is the DNA and not tail that is stained?
Definitely you can not differentiate between tailed or not-tailed by FC, instead using fluorescence microscopy would be the best. Staining cells with FDA will be sufficient for this purposes.
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