Infection with pMMP retrovirus

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Infection

• 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).

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