I am trying to isolate the AM from BAL of mice. I read several articles and protocols. Some of them use cold buffer. Some use prewarmed buffer (37℃). Although the content of the buffer migh be the same, what is the difference between cold and warm buffer for AM isolation?
Hi,
We used Cold buffer to avoid macrophages adhering to the epithelium or sticking to plastic tubes upon harvesting.
Inject idealy cations-free (Mg-and Ca-) PBS containing 0.5 mM EDTA, and placing your buffer tubes on ice. We performed 10 washs to harvest 0.25 - 1 X 10(6) cells per mice.
To collect your macrophages, spin at gentle speed 3500 to 5000 rpm for 5 minutes.
Hi, our group also macrophages collection using cold buffer.
Thus, decrease cell adhesion in the tubes and improves the performance of collection.
Spinning, we use 1500 rpm / 5 min.
Hi,
The rule is to use cold PBS (w/o Mg/Ca) for macrophages in suspension, for example during the isolation procedures where you want to minimize the cell attachment and warm PBS for adherent macrophages when you want to minimize the detachment for example during the washing steps for medium change in the plate.
- Badges
- Science method