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.

Related Protocols
-----------------

- :doc:`retrovirus-production`