I have been harvesting monocyte-derived macrophages from buffalo and cattle fresh whole blood using the same protocol for years and getting good macrophage cultures. Below is the protocol that I use:
1. Dilute the blood 1:1 with HBSS
2. Layer 10ml of diluted blood onto 5ml of Ficoll-Paque
3. Centrifuge at 600xg, 35min, 18°C
4. Take the PBMC, layer onto Percoll (1:1)
5. Centrifuge at 580xg, 15min, 4°C
6. Collect buffy coat (monocyte-rich layer) and incubate with RBC cell lysis for 10min.
7. Centrifuge at 300xg, 15min, 4°C
8. Wash with HBSS
9. Centrifuge at 300xg, 15min, 4°C
10. Culture in RPMI + 1% anti anti + 20% FBS, 5% C02, 37°C for 21 days until maturation into macrophages
However, lately, I have been getting mixed cell populations in my flask, which confuses me. I have included images of the not-so-familiar cell population on day 12. Please advise. Thank you.
Do you use Wright’s staining before culturing cells? Perhaps the harvesting protocol is proper, but you might get contamination later on.
In your paper, Phagocytosis and Intracellular Killing of Pasteurella multocida B:2 by Macrophages: A Comparative Study Between Buffalo and Cattle, you report 600xg, 35 min centrifugation at 4°C, but here you mentioned 18°C. Is this an error, or could this discrepancy be the source of the issue?
Jakub Jagielski Yes, I used Wright stain before culturing the cells. I am tweaking the temperature a bit, as I sometimes notice a higher yield of PBMC at 18°C, but it is not finalised yet. Based on the morphology, I suspect the contamination is fibroblast.
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