I've extracted cells via perfusion with saline (PBS) but would love any advice about whether to use a different form of saline (insect medium, carlson's saline?) and whether I should use an anticoagulant. As I am working with undergrads and not full-timers, it would be ideal if we could fix the cells so we can run the next day on the flow cytometer. Any advice would be great.
Hi Robin,
We just used a protocol to do flow cytometry on mosquito hemocytes. The anticoagulant buffer we used is 60% Schneider medium, 10% FBS, 30% citrate buffer [98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, 41 mM citrate]. After collection in anticoagulant buffer on ice, hemocytes were centrifuged immediately at 2350 x g at 4ºC. Cells were resuspended in 200 μl PBS with 0.1% FBS. 700 μl of 70% ethanol was added drop wise to the cells and incubated for 1 h at RT.
The full protocol can be found at http://jeb.biologists.org/content/early/2013/12/16/jeb.094573.long
Let me know if you have difficulties getting the article, I will be happy to send you a pdf reprint.
Good luck! Cheers, Kristin.
Hi Robin,
My friend (Rafael Patiño) was working with Periplaneta (at University of Valencia, Cavanilles Institute of Biodiversity and Evolution Biology) for several years and maybe he can help you. Please find the e-mail address: [email protected]
I hope it can help you with this.
I have a little experience with Biomphalaria`s hemocytes and we were able to fix them with 4%PFA. With a lower % of PFA, we lost many cells since these are highly adherent cells and it seems they did not fix at all.
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