Infection with pMMP retrovirus
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- Plate GM6914 cells onto 10-cm dishes.
- Grow to ~50% confluency.
- Thaw one aliquot of pMMP viral supernatant (SN) on ice
(may take several hours!)
- Mix 50 ml DMEM/15% FCS with 1 ml 100X polybrene
(result: “2X polybrene medium”).
- Only 5 ml of the 2X polybrene medium is needed per infection,
mix only as much as needed.
- Add 5 ml 2X polybrene medium to 5 ml viral SN.
- Incubate briefly at 37 degrees.
- Rinse GM6914 cells once with PBS.
- Aspirate PBS.
- Add virus/polybrene to cells.
- Incubate at 37 degrees/ 5% CO2 for 4-6 hours.
- Change medium using DMEM/15% FCS.
100X Polybene stock sol’n
- Weigh in 80 mg polybrene (Hexadmethrine bromide, Sigma H-9268).
- Dissolve in 50 ml DMEM.
- Filter through 0.2 um and store frozen (-20 degrees) in single-use aliquots (1 ml).