Viral Production - IMCD Cells

Procedure

Monday

  • Plate cells so that they will be 70% on Tuesday.

Tuesday

  • Transfect Cells.

    • 8 micrograms of PSPAX2

    • 8 micrograms of CMV100 vector

    • 1 microgram of PMD2.G

    • 700 microliters of optimum

    • 20 microliters of lipofectamine LTX

    • Let incubate at room temperature for 30 minutes, and then add dropwise to HEK293T cells with 9 mL of fresh media. Total volume, ~10 mL.

Wednesday

  • Gently aspirate media from HEK293 cells, and gently add 10 mL of fresh media.

  • I usually add it to the side of the dish dropwise since the HEK cells lift off the dish easily.

  • Do whatever you gotta do to have the IMCD cells happy and ready for transduction the next day.

Thursday

  • Gently filter the viral supernatant.

  • Add 10 mL of fresh media to the HEK293T cells.

  • Add 7 mL of fresh media to IMCD cells.

  • Add 10 ul of polybrene (10 mg/mL) to IMCD cells. Mix gently.

  • Add 3 mL of viral supernatant to IMCD cells. Mix gently.

Friday

  • Do the same for the IMCD cells with 72-hour supernatant.

  • Change media for IMCD cells from yesterday. Let sit till Monday.

Saturday

  • Change media for second infection of IMCD cells. Let sit till Monday.

  • Get a strong drink.