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protocolPageIcons_protocols_TJF_2016_6_10-01.png Colony Formation Titering Assay for Lentivirus


Introduction

This protocol can be used to titer lentivirus preparations that confer antibiotic resistance. Determining the titer of your lentiviral preparation allows you to control the multiplicity of infection (MOI) in downstream studies. This protocol was developed for A549 cells but can be adapted to a variety of target cell lines and selection markers. Note this assay requires staining resistant colonies with crystal violet solution and, therefore, the cells cannot be used for later experiments.

Last Upload: June 16, 2016

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Workflow Timeline

Day 0: Seed and Transduce Cells

Day 2: Replace media with fresh media containing selection reagent.

Days 3-14: Change media as needed

Days 14-18: Stain cells and count resistant colonies

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Equipment

  • Biosafety cabinet
  • Pipetman
  • Pipettors
  • Incubator

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Reagents

  • DMEM high glucose
  • L-alanyl-L-glutamine (or alternative stable glutamine)
  • Heat-inactivated FBS
  • Polybrene
  • PBS pH 7.4 without calcium or magnesium (cations can affect the attachment of adherent cells)
  • Microcentrifuge tubes
  • 6-well dishes
  • Pipettes
  • Pipette tips
  • 0.1% Crystal Violet
  • 0.22 μm polyethersulfone filter (for crystal violet solution)
  • 0.45 μm polyethersulfone filter (for viral preps, if prep was not previously filtered)
  • Syringe for filtering
  • Lentivirus Preparation

Reagent Preparation

  1. DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine
    • To a 500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and 11 mL of 200 mM L-alanyl-L-glutamine. Store at 4℃.
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    *Pro-Tip* Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one suitable with your protocols. FBS can be purchased already head inactivated or it can be inactivated in the lab by heating to 56℃ for 30 minutes.

Considerations Before You Start

  • The health of the the target cell line is critical for obtaining accurate titers.
    • Check the cells for mycoplasma regularly
    • Do not over or under-grow your cells.
    • Thaw a new vial of cells after 20-30 passages.

  • Do not add penicillin/streptomycin to the media.
  • Titer will vary between cell lines.
  • It is not recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles.

Procedure

  1. Before beginning a colony formation assay, the dose of antibiotic required to kill your target cell line needs to be empirically determined. Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration required to kill all of the cells. Use this dose for the colony formation assay.
  2. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting 20 μL of 10 mg/mL polybrene into 20 mL media.
  3. Incubate the cells overnight.
  4. If using freshly collected virus, filter through a 0.45 μm polyethersulfone filter to remove cells and debris.

    • Lentiviral titer can decrease during cycles of freeze-thaw. If you are freezing and aliquoting virus it is recommended that you titer from the frozen stock to account for any loss in titer associated with freeze-thaw.
  5. If using frozen virus, rapidly thaw the lentiviral aliquot at 37℃ by agitating in a warm water or bead bath.
  6. Prepare 10-fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene:

    Dilution Volume of Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus Added to Plate (μL) Volume of Cells Added to Plate (μL) Final Viral Dilution
    1:10 100 of Stock Virus 900 150 1,350 1:100
    1:100 100 of 1:10 900 150 1,350 1:1,000
    1:1,000 100 of 1:100 900 150 1,350 1:10,000
    1:10,000 100 of 1:1,000 900 150 1,350 1:100,000
    1:100,000 100 of 1:10,000 900 150 1,350 1:1,000,000
    • Mix the dilutions well
    • Note: the 1:10 dilution can usually be omitted because this dilution typically produces a lawn of cells, which cannot be accurately quantified.
  7. Add 150 μL of a viral dilution to each well (each well gets one dilution)
  8. Seed 1,000 cells into each well of a 6-well dish.
    • Prepare a batch of cells as follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene.
    • Mix well by pipetting or inverting.
    • Aliquot 1.35 mL of the cell suspension into each well of the 6-well dish directly on top of virus. Swirl gently to mix and place dish in incubator.

  9. Incubate for 48-72 h.
  10. Gently aspirate the media from the cells.
  11. Add 1.5 mL of DMEM complete containing the appropriate antibiotic.
  12. Incubate the cells for ~2 weeks. All of the cells in the untransduced (negative) control well should be killed and colonies should be visible.
  13. Every 3-4 days, gently aspirate the media and replace it with fresh DMEM complete containing the appropriate antibiotic.
  14. When the untransduced (negative control) cells have died and colonies are visible in the transduced wells, aspirate the media from the wells.
  15. Gently wash the cells with 1 mL of PBS and aspirate wash.
  16. Filter 0.1% crystal violet solution through a 0.22 μm filter to remove any precipitates.
  17. Stain each well with 1 mL of 0.1% crystal violet for 10 min at room temperature.
  18. Gently remove the stain.
  19. Wash cells 3x with 1 mL of PBS, being careful not to disturb the colonies.
  20. Count the colonies for at least 2 of the dilutions.
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    *Pro-Tip* The higher dilution wells tend to provide more accurate titers as the likelihood of multiple integration events decreases as the dilution increases.
  21. Calculate the transduction units per mL (TU/mL) as follows:

    • TU/mL = number of colonies/total volume in the well (mL) x dilution factor
    • e.g., If the 1:100,000 well has 75 colonies, then there are 75 colonies /1.5 mL x 100,000 or 5,000,000 TU/mL
  22. For a more accurate titer, take the average of multiple dilutions.

Sample Data

Crystal Violet Assay

Figure 1: A549 cells were transduced with the indicated serial dilutions of the lentiviral vector pRosetta. The following day, cells were treated with puromycin. Colonies were allowed to grow in the presence of selective reagent for 12 days with media exchanges every 3-4 days. Colonies were then stained with 0.1% crystal violet and counted.

Colony Formation Assay

Figure 2: A549 cells were transduced with the indicated serial dilutions of the lentiviral vector pHAGE EF1α dCas9-KRAB. The following day, cells were treated with puromycin. Colonies were allowed to grow in the presence of selective reagent for 12 days with media exchanges every 3-4 days. After 12 days of selection, no cells have survived in the untransduced well, while lawns of cells are present in the lower dilutions, and single colonies are visible in the higher dilutions.