Hi everyone! How many cells do you used to collect by bronchoalveolar lavage from male C57BL/6 mouse? I would like do more assays but my cell count is low.
Thank you!
One million macrophages in normal mouse. Lavage 4-6 times with one ml each.
Also, when you sacrifice the mouse use heparin to reduce clotting and tissue retension of the macrophages. You will see the surfactant in your lavage and as it wanes, you will know that your lavage is quantitative. Also, withdraw all air before insufflating the lungs. If you have a problem, send me a video to analyze.
Michael! My assays is developed using C57B/6. I use to wash the lungs with cold PBS. When I wash 6 times, I got one million macrophages, but sometimes I got more cells: 2 or 3 millions. Unfortunately, it does not happen with all the animals that I collect the alveolar macrophages.
I use HBSS or serum free basal media. You will be more consistent if you remove trapped air from the lungs.
Michael, how do you remove trapped air from the lungs? and second, once you recover this cells, and you let them adhere to plastic well plates, how may cells you recover after? Is FCS an issue regarding culturing them? or it doesn't matter? My PhD student says all the cells are detaching after 2 hours culturing..
And one more thing... the heparin concentration you use? (If I understood properly, is diluted in the ice cold PBS, isn't it?)
The heparin is administered IP prior to sacrifice so that during lavage, cappilary breakage does not lead to clots.
Sorry for my terse incomplete answers. Deeply anesthetize the animal with IP phenobarbital or approved drug with heparin. Insufflate the lungs with room temperature or warm serum free media to minimize coagulation. Plate on tissue culture plastic. Macrophages will adhere in less than one hour. Wash and replace with serum free medium.
- Badges
- Science topic