To begin, obtain human mononuclear cells from whole blood. Vortex human FcR blocking reagent, and add the required amount of reagent to the cells. Incubate the tube with cells at four degrees Celsius for five minutes.
Add 20 microliters of CD11b MicroBeads per 10 to the power of seven total cells, and incubate at four degrees Celsius for 20 minutes. Thoroughly disinfect the magnetic stand with 70%ethanol. After incubation, add 10 milliliters of isolation buffer.
Mix, and centrifuge the tube at 300G for 10 minutes at four degrees celsius. Once the stand dries, position the magnetic column on the magnetic stand. After removing the supernatant, add one milliliter of isolation buffer and mix gently with the pipette.
Then load the cell suspension onto the column and wash it three times with three milliliters of isolation buffer each time the column reservoir is empty. Place the column in a 15-milliliter conical tube without touching the magnetic part. After adding five milliliters of isolation buffer, push the plunger into the tube to force the liquid out through the column.
Centrifuge the cell suspension collected in the tube. Aspirate the supernatant and re-suspend the cells in isolation medium. Following cell counting, aliquot 500 microliters of cell suspension into each well of a 24-well plate, and incubate overnight.
To induce microglia-like cells, replace the isolation medium from the seeding of the previous day with an induction medium and incubate the cells for 14 days. The induced microglia-like cells exhibited minute soma bodies and numerous branched collaterals.
