I am working on lung in vivo infections, but I now need to do some in vitro experiments with primary alveolar macrophages. i am using mice with B6 background.
I have established a protocol where I use PBs+0,1 mM EDTA for BAL, 0.8 ml x 5 /per mouse. I pool the BALF from 5-6 mice, spin down 450 g, 10', count and estimate viability and them plate them in 24 well flat bottom tissue culture treated dishes (RPMI + 10% FCS+pen strep)
My obstacles:
- low yield of cells - per mouse I get max 280 000 cells (before plating) - cells are more than 95 % AMs. Do you have any suggestions on how to increase this yield?
- when I plate cells in 24-well dishes, my cells adhere, but tend to adhere more close to the edge of the well (walls of the well), so that in the middle of the dish, confluence is much lower than at the borders...i mix cells after adding them to the plate, of course...We use CytoOne dishes, maybe I should change the plates? Did any of you encounter this issue with cells grouping more closely to the walls of the well?
Thanks a lot for any suggestions!
You can please refer to the links below to obtain certain interesting informations:-
1. http://www.macrophages.com/isolation-mouse-alveolar-macrophages
2. http://www.jove.com/video/3883/harvesting-murine-alveolar-macrophages-evaluating-cellular-activation
http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20X%5Bauth%5D
this paper may help you.
well,I've just worked on rat in my researches ,and the protocol of isolation of primary alveolar macrophages in rat which I used is as the same as mouse.But why u don't use rat instead of mouse to solve the problem of number of cells?
There is a good alternative to solve this problem. Please see attached article. I also did a very vigorous validation of MPI macrophages and similar to the authors found that these cells are very similar to alveolar macrophages. Typically I can get large number of MPI macrophages in two weeks (I am working with just born mice, which is slightly different from published protocol). At the moment I made MPI macrophages from all our KO mice and trying to do KI with these cells. Hope this will help you.
I have seen/heard about different methods of BALF collection -- keep the lungs in the mouse with the diaphragm cut/torso open, keep the lungs in the mouse with diaphragm intact (as described in Sandip's first link), or take the lungs and heart out of the mouse while collecting and massage the lungs to promote mac detachment.
Does anyone have experience with pros and cons of these methods, or comparative yields?
I prefer the previous method (the method I used)because it's safe and not risky to lose the alive cells due to thoroughly strile situation.
Interesting point. I will be culturing these macs in suspension for a few days, so sterility is important! Thanks!
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