Changes

Murine macrophages and macrophage–like cell lines such as RAW 264.7 adhere to tissue culture–grade plastic through cation–dependent integrin receptors and other cation-independent receptors, predominantly the murine scavenger receptors (MSRs) (Fraser, Hughes, and Gordon. [1993] Nature 364[6435]:343-346). In order to reduce adhesion during routine culture, the RAW 264.7 cells are grown on sterile non-tissue–grade plastic (ultra-dish Petri dishes). Adherence of macrophages to these plates is mediated by αMβ2 (CR3) integrins (Rosen and Gordon. [1987] J. Exp. Med.166[6]:1685-1701), and this interaction is readily reversed by using cation chelators such as EDTA. This adhesive interaction is also sufficiently weak that cells may be detached by the sheer force of media flowing over the cells.

Note: macrophages are extremely sensitive to lipopolysaccharide (LPS) endotoxin from Gram-negative bacteria. LPS has major effects on macrophage phenotype and function, including adhesion. All solutions, buffers, and media should be made with sterile, endotoxin-tested, distilled, deionized water.

==Reagents and Materials==

*Hemacytometer: Fisher Scientific; catalog no. 02-671-5

#Transfer RAW 264.7 growth medium 1 (RAWGM1) to a T150 flask (100 ml of growth medium per flask).

#Place flask in incubator at 37 °C with a 5% CO2 atmosphere for at least 30 min to equilibrate the medium. (Note: if the medium is clearly pink and likely has a pH >7.6, remake the medium; all manipulations and additions to cells are done with sterile tissue culture technique.)