Fluorescence Titering Assay for Lentivirus

Introduction

This protocol can be used to titer lentiviral preparations that carry a fluorescence marker. Determining the titer of your lentiviral vector allows you to control the multiplicity of infection (MOI) in downstream studies. This protocol was developed for 293T cells but can be adapted to a variety of target cell lines and selection markers. Note, because this protocol uses microscopy to determine the percentage of fluorescent cells, these cells can still be used in downstream applications.

Safety Warnings

Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations.

Workflow Timeline

Day 0:

Seed 293T cells

Day 1:

Transduce cells

Day 2 (am):

Remove media, replace with fresh media

Day 4–5:

Count fluorescent cells

Equipment

• Class II, Type A2 Biological Safety Cabinet
• 0.5–10 µL single channel pipette
• 2–20 µL single channel pipette
• 20–200 µL single channel pipette
• 200–1000 µL single channel pipette
• Ice bucket
• CO₂ incubator
• Pipet controller
• Hazardous waste container
• Inverted fluorescence microscope

Reagents

• DMEM high glucose, Corning 10-013-CV
• L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI)
• Heat-inactivated FBS
• Polybrene (10mg/mL), Millipore TR-1003-G
• 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells)
• 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered)
• Microcentrifuge tubes, Neptune 3745.X
• 6-well tissue culture treated dish, Corning 3516
• 15 mL conical tubes, VWR 21008-216
• Lentivirus preparation

Reagent Preparation

1. DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such as glutaGRO)
   • To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C.
   Pro-Tips
   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 heat-inactivated or it can be inactivated in the lab by heating to 56 °C for 30 min.

Considerations Before You Start

• The health of 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, as it can affect viability and transduction efficiency.
• Titer will vary between cell lines.
• It is not recommended to subject lentiviral supernatants to multiple freeze-thaw cycles.

Procedure

1. Seed 75,000 cells into each well of a 6-well dish.
   • Prepare a batch of cells as follows: Dilute 525,000 cells into 14 mL of DMEM complete.
   • Mix well by pipetting or inverting.
   • Aliquot 2 mL of cell suspension into each well of the 6-well dish.
2. Incubate the cells overnight.
3. 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.
4. If using frozen virus, rapidly thaw the lentiviral aliquot on ice prior to use.
5. Prepare dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol was developed using low titer lentiviral vectors (10⁵ TU/mL). If you anticipate that your viral stock will be higher titer, consider additional dilutions.

   Dilution	Volume of Lentivirus Stock (μL)	Volume of DMEM complete (μL)	Volume of 10mg/mL polybrene (μL)
   1:10	150	1348.5	1.5
   1:25	60	1438.5	1.5
   1:50	30	1468.5	1.5
   1:75	20	1478.5	1.5
   1:100	15	1483.5	1.5
   Untransduced	0	1498.5	1.5

   • Mix the dilutions well
6. Gently aspirate media from the cells.
7. Add 1.5 mL of a viral dilution to each well (each well gets one dilution with one well remaining as the untransduced control).
8. Count the cells in the remaining well, a cell count is required for calculating the titer.
9. Incubate for 48–72 h.
10. Gently aspirate media and replace with 1 mL of PBS.
11. Calculate the fraction of fluorescent-positive cells in each well.
    • When calculating titer, only consider wells with less than 40% fluorescent cells. Titering methods assume one integration event per cell. When the percentage exceeds 40% you risk counting cells with multiple integration events leading to underestimation of the true titer.
12. Calculate the transduction units per mL (TU/mL) using either the dilution factors (method 1) or the volume of virus (method 2):

    Method 1	Method 2
    $$T = {N*F*D\over V_T}$$ Where: T = Titer, TU/mL N = Number of cells transduced F = Fraction of cells with fluorescence D = Dilution Factor VT = Transduction Volume, mL For example: If 150,000 cells were transduced in the 1:100 well, resulting in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent x 100)/(1.5 mL) = 2.5x106 TU/mL	$$T = {N*F\over V_V}$$ Where: T = Titer, TU/mL N = Number of cells transduced F = Fraction of cells with fluorescence VV = Virus Volume, mL For example: If 15 µL of virus added to 150,000 cells resulted in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent)/(0.015 mL) = 2.5x106 TU/mL

13. For a more accurate titer, take the average of multiple dilutions.

Sample Data

Figure 1: 293T cells were transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry. 72 h post transduction, cells were assayed for mCherry expression using fluorescence microscopy.

Last reviewed on: September 9, 2023