细胞世界

10-15 min maximum will do, at 37oC. But not Sigma kit. Sigma kit is an hour.

Yes i do.~~~~~~~~~~·

(1) when you plate RAW264.7 cells, you add sRANKL. (same time).
(2) cells will stop to proliferate when sRANKL is added, so your problem is not a problem.
(3) the reason you have this problem (cells still dividing when RANKL added) is: your raw264.7 cells are too old (i mean the generation).

2-10 million cells per 60-mm Petri-dish. If you use small wells in a plate, certainly cell number has to be reduced.

There is no any difference with/without M-CSF, but M-CSF is not necessary.

Passage <20 should be ok for osteoclasteogenesis.

I do not like that kit for cells labelled as brown but not dark red (more pretty).