Titer Test

K562 Titer Test


For Assessing Lentiviral Titer

Important Notes

  1. This protocol is optimized on K562s to measure lentiviral titer; however, it can be adapted to other cell lines to assess transduction efficiency.
  2. BL2+ practices must be maintained until at least after the expansion to 15cm dishes on Day One.
  3. Make sure to plate an untransduced well of cells as a control.
  4. Spinfection can be done in late PM with expansion to 15cm dishes on day 2 early AM (16h later). Subsequent steps would be performed on days 3-7.


    5. Day One
    AM BL2+
    1. Spin K562 cells at 1200rpm for 4min. Aspirate media. Resuspend in RPMI + 10% FBS + P/S.
    2. Count cells. Calculate total cell number: you will need 5M per virus volume + 5M for untransduced control, at a density of 5M/ml.
    3. If you have enough cells, remove virus from -80C freezer and thaw at 37C in a water bath.
    4. Make cell mixture at 5M cells/ml (remember to make a little extra).
    5. When virus is thawed, add polybrene to 2X to cells (final concentration for K562s = 10ug/ml; 2X = 20ug/ml). Pipette up and down to mix well.
    6. Seed 1ml of K562 mixture per virus volume into wells of a 6-well plate.
    7. Add the following volumes of virus: 1ml, 0.5ml, 0.25ml, 0.125ml; pipet up and down to mix.
    8. Add media to 2ml final volume to all wells (including untransduced); pipet up and down to mix.
    9. Check the volume in balance plate wells - all should have 2 ml for balance. Wrap the 6-well plate in a Kimwipe and place in sealed centrifuge bucket.
    10. Spin 45min 2200rpm 37C. Remove from centrifuge and place in incubator overnight.
      NOTE: if the cell line is new, keep one plate without spinfection to see how cells tolerate it.

    PM BL2+
    1. Pipet cells into 15ml or 50ml conical tubes (one tube/well).
    2. Spin 4min 1200rpm. Aspirate media.
    3. Resuspend cells in 20ml RPMI + 10% FBS + P/S. Plate in 15cm dishes.

    Day Two
    1. Allow cells to recover.

    Day Three
    BL2+
    1. Pipette cells into 50ml conical tubes (one tube/15cm dish).
    2. Spin 4min 1200rpm. Aspirate media.
    3. Resuspend cells in 10ml RPMI + 10% FBS + P/S. Count cells.
    4. Plate cells at 200K cells/ml in 20ml (5M cells total) in RPMI + 10% FBS + P/S. Add puromycin to 3ug/ml final.
      Seed two control plates of untransduced cells: unselected and puromycin selected.

    Days Four and Five
    1. Puromycin selection.

    Day Six
    1. Pipette cells into 50ml conical tubes (one tube/dish).
    2. Spin 4min 1200rpm. Aspirate media.
    3. Resuspend in 10ml RPMI + 10% FBS + P/S. Count cells.
    4. Verify that selection is complete by observing untransduced selected cells and counting. Calculate % infection of all virus volumes, relative to untransduced unselected cells.

    For titer test on cell line to be screened: modify transduction parameters as necessary on days one through three.
    At day 6, also calculate the approximate cell number and volume of virus necessary for 1000X coverage of the library on the day of transduction (day one).

    If your library contains 100,000 sgRNAs and if % infection was 50% with 500ul virus on 5M cells:
    100M cells (1000X coverage) / 0.5 (% transduction efficiency) = 200M cells
    200M cells * 0.5ml virus / 5M cells = 20ml virus