I tried to isolate myeloid cells (microglia and macrophage) from adult mouse brain. Then I sorted CD11b and CD45 double positive cells through flowcytometer. However, the viability of the sorted cells is not high enough for my following assay. I want more than 90% live cells after FACS.
In my experiment, I used PBS (pH 7.4) containing 0.04% BSA as FACS buffer and collecting buffer after sorting. I used 70um and 85um nozzle for FACS and it doesn't make any difference.
I'm asking what buffer you would recommend for FACS and collecting buffer after sorting? What nozzle size you used for FACS. Also please feel free to post or direct me to your protocols that work for your myeloid cell FACS.
Thank you so much!
Hello
These Links will help you
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354492/
http://www.pnas.org/content/113/12/E1738.full.pdf
https://link.springer.com/protocol/10.1007/978-1-4939-1212-4_2#page-1
http://www.jimmunol.org/content/166/4/2717
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980480/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145600/
Good Luck
Hello Dr. Hongtian Stanley Yang ,
I think, you need to increase your BSA percentage from 0.04% to 1-2% . And i use 100um nozzle.
For details, kindly knock me at [email protected]
Sincerely,
Babu
Yes, i will agree with Nicole Amberg . You can add 10% FBS . But depending on your experimental design, you can add 0.1% sodium azide too.
Up your BSA concentration Stanley. For our retinal and brain microglia we did 2% BSA in HBSS. We also used DNase II from Worthington Biochem. This significantly improved our yield. In our final experiments, we only used >95% viable samples. Pop over to my office if you want to chat!
Pete
Thank you all for your generous share. Will definitely increase BSA concentration.
Hongtian Stanley Yang or anyone else: Could you, please, share your protocol with me? I'm planning to perform microglia sorting but I cound't find a good detailed protocol.
Thank you in advance.
Natalia
Natália Ferreira Mendes
This protocol will help you!
Article Isolation and Culture of Microglia
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