To begin, seed 20 million neonate mouse bone marrow cells per T75 flask in 10 milliliters of complete DMEM containing 2.5 milliliters of 10%L929 cell supernatant. Incubate the culture flask at 37 degrees Celsius with 5%carbon dioxide for five days. On day three of differentiation, add two milliliters of L929 conditioned media into the flask.
Under an inverted microscope with 20x magnification, daily observe the cells. To harvest the macrophages on day five, aspirate the differentiation media from the flask. Wash the cells with five milliliters of PBS to remove non-adherent cells and serum proteins.
Add five milliliters of 0.05%trypsin EDTA to the flask and incubate at 37 degrees Celsius incubator with 5%carbon dioxide. After five minutes, add five milliliters of DMEM and pipette to detach the cells. Centrifuge the cell suspension at 350 g for five minutes.
Dissociate the cell palate in one milliliter of complete DMEM. Count the cells on an automated cell counter. In the presence of L929 cell supernatant, bone marrow cells were differentiated into macrophages within five days.
Spindle-shaped cell formation was observed from the second day with nearly half displaying this morphology by day three and a majority by day five.
