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• heat-inactivated horse serum to a final concentration of 5%
• heat-inactivated fetal bovine serum to a final concentration of 10%

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Transfer all medium and floating cells from flask to a centrifuge tube.
2. Briefly rinse the cell layers with Ca⁺⁺/Mg⁺⁺ free Dulbecco's phosphate-buffered saline (D-PBS). Add rinse solution to the cells in centrifuge tube in step 1.
3. Add 5.0 ml of phosphate buffered saline containing 1 mM EDTA, 1 mM EGTA and 1 mg/mL glucose solution to flask to detach cells. Observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. After the cells have detached, transfer them to the centrifuge tube in step 1.
5. Centrifuge the cells at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
6. Resuspend the cell pellet in fresh growth medium.
7. Add appropriate aliquots of cell suspension to new culture vessels.
8. Place culture vessels in incubators at 37°C.

Medium renewal: every 2 to 3 days

Nagamoto-Combs K, et al. A novel cell line from spontaneously immortalized murine microglia. J. Neurosci. Methods 233: 187-198, 2014. PubMed: 24975292