I have been trying to analyze Kupffer cell from C57BL/6 mouse liver by flow cytometry following standard protocols:
1. Liver harvest (I tried both before and after perfusion)
2. Liver chopping with dorco razor blade.
3. Enzyme digestion (Collagenase D and DNase I, 0.5~1 hour)
4. Cell strainer (40 um)
5. Centrifugation (50 g, 1~3 min)
6. Obtain supernatant
7. Centrifugation (400 g, 5 min)
8. RBC lysis
OR
(1~4 same)
5. Centrifugation (300 g, 5 min)
6. RBC lysis
I also tried using Percoll gradient (33% or 30/70%), but I couldn't see CD11b-int and F4/80-hi Kupffer cell population in my flow cytometry data. I could only identify CD11b-hi F4/80-int(?) population which, I am assuming, is Monocyte-derived macrophages.
Is there anything I could have possibly done wrong, or anything I should do?
Thanks!
Hi,
This is a very simple and effective method of Kupffer cell isolation:
Article Kupffer Cell Isolation for Nanoparticle Toxicity Testing
Good luck!
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