6.1: Performing Lentiviral Transduction and Flow Cytometry on NIH3T3 Cells
As you proceed through this set of protocols and experiments, be sure to enter all your work into your
notebook!
Objectives
Successfully perform lentiviral transduction to generate stable GFP-positive NIH3T3 cells
ALERT!
For your own safety, NO MANIPULATIONS of lentivirus or lentivirus-containing samples can occur
outside the 3 biosafety cabinets. You are working with BSL2 materials in this experiment.
The nature of the protocol requires some activities to take place on TUESDAY and THURSDAY.
Vocabulary
Lentivirus, transduction, stable
Outline
Distribute cells to wells of a 96-well plate; transduce with lentivirus; remove lentivirus solution; allow
recovery; replate cells and select for puromycin resistance; assess by flow cytometry for GFP expression
Materials
sterile
Trypsin, PBS, 3T3 growth medium
Puromycin (10mg/mL)
one 96-well plate
one 24-well plate
Lentiviral particles containing the GFP transgene construct (Sigma SHC003V)
Hexadimethrine bromide (Polybrene), 2mg/ml stock
1.5ml eppendorf tube
serological pipettes
plastic pipette tips
non-sterile
37C water bath and rack (as necessary), maintained with antimicrobial additive
Dry ice
Trypan blue solution, hemocytometer
fully-stocked biosafety cabinet
kimwipes for swabbing and 70% ethanol
DAY 1 (Monday)
Protocol
PREPARATION
1. Prepare the hood and collect all materials
2. Label the new materials as necessary given the instructions that follow.
WASH, TRYPSINIZE AND COUNT ADHERENT CELLS
3. Aspirate the old media, wash with PBS, trypsinize and mechanically disperse your cells.
4. Using a hemocytometer, calculate the concentration of live cells in your cell suspension stock.
REPLATE AT SPECIFIED CELL DENSITY
5. Seed cells into 6 wells of a 96-well plate at a starting confluence of 40%. Only two of the wells
will be used, four additional wells are seeded as back up. Our goal is to reach 70-80% confluence
on the day of transduction (tomorrow). If you are unable to predict from your own prior
experiments how many cells to seed, try 104, 2x104, 3x104.
a. Well of a 96-well plate at:
(your predicted seed number)
b. Well of a 96-well plate at:
(your predicted seed number)
c. Well of a 96-well plate at:
(half your predicted seed number)
d. Well of a 96-well plate at:
(half your predicted seed number)
e. Well of a 96-well plate at:
(double your predicted seed number)
f.
(double your predicted seed number)
Well of a 96-well plate at:
6. Place the fresh plate in the 37C incubator.
7. Schedule a time with the instructor for transduction tomorrow.
DAY 2 (Tuesday)
Protocol
TRANSDUCTION
1. Examine your plate to determine which seed condition is optimal. Return your plate to the 37C
incubator until ready to transduce.
2. Obtain a sterile microcentrifuge tube containing 500uL of growth medium containing a final
concentration of 8ug/mL Polybrene.
3. Virus should be stored on dry ice until needed. Thaw virus at room temperature for 2-3min.
4. Aspirate the well for transduction, add 110ul growth medium containing polybrene.
5. Add 10uL of the solution containing the lentiviral particles to the transduction well. Gently
agitate plate to evenly mix.
Note: ADD VIRUS DIRECTLY TO THE SOLUTION, NOT TO THE PLASTIC WALL OF THE WELL.
6. Replace the medium of two control wells with fresh medium (no virus, +polybrene).
7. Return the plate to the 37C incubator.
DAY 3 (Wednesday)
Protocol
RECOVERY AFTER TRANSDUCTION
1. Aspirate the medium containing lentiviral particles from the well, add 100uL PBS, aspirate.
2. Treat with 100uL trypsin and transfer all transduced cells to one individual well of a 24-well
plate containing 1mL prewarmed growth medium (no virus, no polybrene).
a. Do not vigorously disperse the cells in the well of the 96-well plate.
b. Set the pipettor to 200ul and gather the cells (in the 100uL of trypsin solution) all in one
pipetting to the new well.
c. Gently disperse the cells in the new well
3. Repeat steps 1 and 2 for the cells from the non-transduced control wells.
4. Return the new plate to the 37C incubator.
5. Make an appropriate volume of growth medium containing puromycin for use tomorrow and
Friday.
DAY 4 (Thursday)
Protocol
PUROMYCIN SELECTION
1. Replace the medium for the transduced cells and one of the non-transduced control wells with
1ml of growth medium containing puromycin at the concentration determined from the kill
curve experiment.
2. Provide growth medium without puromycin to the second control well.
3. Return the plates to the 37C incubator.
DAY 5 (Friday)
Protocol
MEDIA EXCHANGE
1. Observe your cells under the microscope.
2. Replace the medium with fresh medium containing puromycin (or not for the 2nd control).
a. It is likely that there will be many “floater” cells that have been killed by the puromycin.
Aspiration of the old medium should be sufficient to remove most dead cells.
3. Maintain the cells in puromycin containing medium. Refresh medium every 2-4 days or split the
cells to a larger plate as necessary. If the cells are approaching confluence today, split them into
a single 6cm dish.
DAY 8 (Monday)
Protocol
FLOW CYTOMETRY
1. Observe your cells under the microscope.
2. Aspirate, wash with PBS, aspirate and trypsinize your cells from the 24 well plate using 200uL
trypsin.
a. Control +puromycin should contain no viable cells and thus will not be counted
b. Control – puromycin should contain viable cells for counting
c. Transduced +puromycin should contain viable cells for counting
3. Once the cells have released from the plate, add 200uL growth medium to inactivate the trypsin.
4. Transfer the entire volume of cells to separate microcentrifuge tubes and spin for 5 minutes at
1000xg.
5. Aspirate the solutions taking care to avoid disturbing the cell pellets.
6. Resuspend the cells in 500uL PBS and transfer them to separate polypropylene conical tubes for
flow cytometry.
7. Await further instruction for sample processing on the flow cytometer.
Questions:
1. What percent of your transduced and non-transduced control cells are GFP-positive? How do
these percentages compare to your previous observations with transient transfection?
2. Why is lentiviral transduction expected to have higher efficiency than transient transfection
methods?
3. Do all GFP+ cells exhibit the same amount of GFP expression? If not, why might they differ?