Encyclopedia of Experiments: Immunology
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Seed 10 million cells per 10 milliliters in a 10-centimeter culture dish, and add M-CSF to the cells. Incubate the culture at 37 degrees Celsius, 5% carbon dioxide for 3 days. Remove the medium after three days, and wash the cells twice, vigorously, with 10 milliliters of PBS to remove non-adherent cells.
Add 5 milliliters of room temperature 0.02% trypsin-EDTA in PBS, and incubate at 37 degrees Celsius, 5% carbon dioxide for 5 minutes. Thoroughly pipette the cells to detach them. Observe the culture under a microscope to make sure the cells have been detached and appear rounded and floating in media.
When the cells have been detached, inactivate the reaction by adding 5 milliliters of alpha-MEM. Collect the cells into a 50-milliliter conical tube and centrifuge at 300 x g for 5 minutes. After discarding the supernatant, wash with 5 milliliters of alpha-MEM and centrifuge again at 300 x g for 5 minutes. Resuspend the pellet in 10 milliliters of alpha-MEM.
Seed 1 million cells per 10 milliliters in a 10-centimeter culture dish, and add M-CSF. Incubate the culture at 37 degrees Celsius, 5% carbon dioxide for 3 days. Harvest the attached cells, which represent BMMs as osteoclast precursors after 3 days. Seed BMMs at 50,000 cells per 200 microliters of alpha-MEM in a 96-well plate, per well.
To each well, add desired stimuli, and then add M-CSF for a final concentration of 100 nanograms per milliliter. Add anti-c-fms antibody to each well. Incubate the plate at 37 degrees Celsius, 5% carbon dioxide for 4 days, while changing media every other day for 4 days, and then proceed with staining and assays as described in the manuscript.
