I recently did isolation of macrophage/ primary cell from chicken lung using histopaque 1119 separation. Unfortunately, I didnt manage to get enough buffy coat/ cloudy layer .. i only managed to get very little/ minute quantity of cloudy layer for some of the isolation and the rest i didnt get the cloudy layer/ buffy coat at all.
The protocol that i used is as below:
The chicken lungs were dissected out from the animals and rinsed multiple times in cold Hank’s balanced salt solution (HBSS) to ensure that they were free of blood. Using sterile scalpel and forceps, the lungs were minced to approximately 5 mm fragments and soaked in 1000U/mL collagenase type I solution for 30 minutes at 37º C. Dispersed cells and tissue fragments were separated from larger pieces using a 40 um cell strainer. The filtered lung cells were pelleted at 233g for 10 minutes (4º C), followed by re-suspension in HBSS and carefully layered onto 4 mL Histopaque 1119 in a 15 mL conical tube at room temperature (with a 1:1 ratio of cell suspension). The layered cells were spun 40 minutes at 400 g at 20C. The cloudy layer, rich in mononuclear cells, was collected and pelleted, washed with HBSS, and the cells were suspended in PBS (Phosphate Buffer Saline) and the cells were counted using a haematocytometer.
anyone can help me on this? or any recommendation?
Dear Suwaibah
This paper was performed on Isolation of macrophages from rat lung. however, I think this paper can help you:
Lavnikova N, Prokhorova S, Helyar L, Laskin DL. Isolation and partial characterization of subpopulations of alveolar macrophages, granulocytes, and highly enriched interstitial macrophages from rat lung. American journal of respiratory cell and molecular biology. 1993 Apr 1;8:384-.
Best regards
Z.Shakeran
you can find some information in the link below:
Article Infections with Avian Pathogenic and Fecal Escherichia coli ...
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue