Transduction of Cultured Cells with Recombinant Lentiviral Particles Protocol

Experiment Summary

This protocol describes the transduction of cultured cells with recombinant lentivirus expressing fluorescent markers.

Materials and Reagents

Target cell growth medium
100X penicillin/streptomycin solution
Dulbecco's Phosphate Buffered Saline
Trypsin
Polybrene

Equipment

Biosafety cabinet
Humidified tissue culture incubator with 5% CO₂
Tissue culture dishes
100mm plates
150mm plates
6-well plates
OR 12-well plates
Vacuum-driven bottle filter with 0.2 μm pore
Pipetaid
Serological pipettes
50ml conical tubes
15ml conical tubes
5ml polypropylene round bottom tube for flow cytometry
Tabletop centrifuge
Pipettes
Filter tips
Hemocytometer or automated cell counting device
Millex-HV Syringe Filter Unit, 0.45 μm
Millex-HV Syringe Filter Unit, 0.22 μm
10-ml syringe only
BioExpress GM Series Balance

Procedure

Day 0: Prepare Lentiviral Transduction Plates

Plate cells for transduction (Plate at a density that will reach 80% confluency in ~24 hrs (time of transduction).)
Incubate cells in a humidified incubator at 37 °C with 5% CO₂ for 24 hrs.

Day 1: Determine Cell Density & Transduce Cells

Determine cell density.
a) Warm appropriate cell culture medium and DPBS in 37°C water bath, and warm trypsin at room temperature.
b) Aspirate cell culture media of designated counting wells.
c) Rinse aspirated wells with DPBS.
d) 12-well plate: Add 1 ml DPBS / well.
e) Aspirate PBS from each well and add trypsin. Incubate for 7 minutes at 37 °C.
f) 12-well plate: Add 200 uL trypsin / well.
g) Neutralize trypsin by the addition of culture media to each well. Triturate each cell suspension to break up cell clumps. Transfer each cell suspension to a separate labeled 15 ml tube/well.
h) 12-well plate: Add 1 ml culture media / well.
i) Rinse each well with additional culture media for collection of remaining cells. Transfer to matched 15 ml tube. Vortex to mix cell suspension.
g) 12-well plate: Rinse with 1 ml of culture media / well.
k) Add an appropriate amount of DPBS to a new Eppendorf tube for each cell suspension to be counted. Add appropriate amount of each cell suspension to matched Eppendorf tube to make a 1:1 dilution.
l) 12-well plate: Mix 50 μl DPBS with 50 μl cell suspension.
m) Vortex each cell-PBS mix and count via hemocytometer to calculate cell number / well.
Transduce cells with recombinant lentivirus.
a) Make stock solution of polybrene.
b) Make optimized polybrene dilution from 10 mg/ml stock in appropriate amount of growth medium in 15 ml conical tube. Mix well by vortexing.
c) Thaw aliquot(s) of recombinant lentiviral particles at room temperature.
d) Make aliquots of growth medium with X mg/ml polybrene for each well to be transduced in 50 ml conical tubes.
e) Add optimized volume of viral particles to each polybrene dilution aliquot designated for viral transduction. Mix well by vortexing.
f) Remove plates designated for viral transduction from incubator and aspirate media from wells.

Day 2: Replace growth media

In the morning (~9:00 AM) aspirate the transduction medium into a flask containing bleach.
Rinse wells with DPBS and aspirate the DPBS. Repeat twice for a total of three DPBS washes.
a) 12-well plate:1 ml PBS / well.
Add fresh growth medium to each plate of transduced cells.
b) 12-well plate:1 ml of growth media / well.
Incubate cells in a humidified incubator at 37 °C with 5% CO₂ for 48 hours.

Day 3: Flow Cytometry & Microscope Verification

Visualize fluorescent cells and/or quantify the percentage of fluorescent-positive cells by flow cytometry.

* For research use only. Not intended for any clinical use.

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Transduction of Cultured Cells with Recombinant Lentiviral Particles Protocol

Experiment Summary

Materials and Reagents

Equipment

Procedure

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