iPSC Transduction
Ali Albalakhi, Ning Xia
Abstract
This is the iPSC transduction protocol.
Attachments
Steps
Begin transduction with cell line 7026
Day 1:
Split iPS cells into single cells in complete medium with RVC. (500µl per well)
Seed cells at 5.0X104 cells in glass-bottom 24-well plates
Day 2: Calculations are for 24-well plates. (If using a 6 well plate, then seed at 1x105 cells per well, 850µL of medium the next day with 150µL)
Virus infection:
Remove the culture medium from cells, add ( 300µL) fresh medium without RVC into each well
Add 45µl virus and 1µg/ml polybrene into cells and mix it. Incubate in 37 degree, 5% CO2.
| A | B | C | D |
|---|---|---|---|
| PLVX mCherry HLA-A2 | PLVX IRES-Puro HLA-A2 | pLoc Mlana |
Add Polybrene into the infected well to make the final concentration to be 1ug/ml.
Stock Polybrene (10mg/mL)
Make a 100x dilution to make a working concentration of 100µg/mL
(10mg/mL) (Vstock) = (100µg/mL)(0.5mL)
Vstock= 5µL of stock into 495µL of DPBS (-)(-)
(100µg/mL) (Vstock) = (1µg/mL)(0.3mL) --> Vstock= 3µL of working concentration per 300µL of medium added
Day 3:
Monitor the cells for the next 48 hours
Day 4 (or 48 hours later):
Check IRES-GFP or HLA-A2’s expression at least 48 hours after the transduction.
For antibiotic-based selection, the cells will be maintained to reform colonies and subsequently grown in medium with 1µg/mL of Puromycin to initiate selection
