An In Vitro Transduction-Induced Myelination Assay for Oligodendrocyte Precursor Cells
Transcript
For this protocol, have primary oligodendrocyte precursor cells, OPCs, cultured in 10-centimeter plates with 10 milliliters of SATO media containing the following growth factors — CNTF, PDGF, NT3, and Forskolin. The cells must have been cultured for 24 hours under 8% carbon dioxide.
Begin with aspirating the media and remove it completely. Then, replace it with 10 milliliters of the same media with the growth factors. Infect your OPCs with lentivirus, expressing the protein of interest by adding the required volume of virus dropwise into the media.
Also, in a separate OPC plate, mix the control lentivirus that expresses only GFP into the media. Culture the cells for another 48 hours, then collect the OPCs from the plates for seeding onto the cultured DRG neurons.
First, rinse each plate with 8 milliliters of EBSS twice. Make up 1 milliliter of trypsin-EDTA 0.05% with 9 milliliters of warm EBSS. Add 5 milliliters of the diluted trypsin to each plate. Incubate at 37 degrees Celsius for up to two minutes. Neutralize the trypsin by adding 5 milliliters of 30% FBS in EBSS.
Then, using a 10-milliliter pipette, wash the OPCs from the plate's surface. Turn the plate by a quarter-turn each time to collect all the cells. Transfer the cell suspension to a 15-milliliter tube. Centrifuge the cells at room temperature for 15 minutes at 180 g's.
Now, aspirate the supernatant and resuspend the cell pellet in a milliliter of warm SATO media without growth factors, and count the cells. Next, prepare the DRG neurons that are cultured on the 22-millimeter coverslips by completely aspirating the media from their plate.
Then, gently seed 200,000 OPCs, dropwise, to each coverslip. The seeding volume must be less than 200 microliters per coverslip. Let the cells settle without moving the plate. After 10 minutes, top each well with a milliliter of warm SATO media. Now replate the remaining sister OPCs with SATO media containing growth factors. They can be used later to verify expression of the protein of interest.
