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Lentivirus Protocol:
DAY 1
CELL GROWTH:
HEK293T (from CORE-T. Low passage, high viability cells. Cells must be very healthy for good transfection and virus production) DMEM+10% FBS 150cm^2 flask
1)Grow HEK293T cells in DMEM+10% serum. Thaw cells, wash off media, add ~10-15 million cells to 20mL to 150mm^2 flask
DAY 2
Trypsin PBS DMEM+10% FBS 100x20mm plates
1)Plate 2-3 million cells in 10mL DMEM+10% serum per 100mm petri dish and grow overnight. Transfect at 60-70% confluency. 2 dishes per transfection. ----To remove cells from 150cm^2 flask: Aspirate + discard media, wash in 8-10mL PBS, aspirate + discard PBS, add 2mL Trypsin (just enough to cover the cells), incubate 2-3 minutes, knock cells loose. Add 10mL 10% DMEM (to a concentration of ~2million cells/mL) which also deactivates Trypsin. Plate cells or freeze down at 10-15 million cells/2mL. Final volume in 150cm^2 flask is 20mL
DAY 3
TRANSFECTION:
H 2 O (autoclaved and .22 filter sterilized) 20 μg of DNA per transfection -3 μg Rev, 5 μg gp, 1.5 μg VSV G make up 9.5 μg, the rest is transfer vector 2M CaCl 2 2xHBS 50mM Chloroquine
1)Check the confluency of the cells. 80-90%.
2)Prepare DNA for transfection, ALL REAGENTS AT ROOM TEMPERATURE Rev ....................... 3μg 1.8μg/μL 1.6μL (totals are for Abhik’s DNA) gp ......................... 5μg 2.3μg/μL 2.17μL VSV G ................. 1.5μg .5μg/μL 3 μL Transfer vector ..... 10.5μg TOTAL ................ 20μg 6.77μL
Make up the volume with H2O (autoclaved, .22 μm filtered sterilized) up to a final volume of 438 μL Add 62 μL 2M CaCl 2 Add 500 μL 2xHBS slowly, mix by bubbling from the bottom of the tube.
Incubate in the hood at room temperature for 30 minutes, the DNA should precipitate
3)5 minutes prior to transfection, Add 6 μL of 50mM Chloroquine into the 10mL of plated media (to a concentration of 25μM). Don’t leave Chloroquine on the cells for more than 24 hours due to cytotoxicity.
4)Pipette up and down and carefully add (don’t add too quickly or you’ll wash the cells off the plate!) the 1mL of precipitated DNA to cells and incubate overnight in 3% CO 2 incubator (10-12 up to 24 hours from adding chloroquine. 24 Hours has been done and is fine. I did 22 and it seemed a little bit long…).
DAY 4
DMEM+5% FBS
1M HEPES
0.5M NaBu
1)Warm 10mL per dish of DMEM + 5% FBS + 10mM HEPES + 10mM Sodium Butyrate to 37°C. [for me, 39mL of DMEM + 5% FBS + 800 μL 0.5M Sodium Butyrate + 400 μL of .22μm filtered 1M HEPES] 2)Check cells under fluorescent microscope for %transfection. Take pictures. 3)Aspirate/discard the transfection media 4)Add 10mL of the warmed media to each 100mm^2 plate. Use the 25mL stripette, it’s end is wider so you’re less likely to wash the cells off. 5)Incubate 6-10 hours (in 10% CO 2 if possible). Don’t go over 10 hours because Sodium Butyrate is toxic to cells 6)Aspirate/discard the media, add 10mL of DMEM + 5% FBS + 10mM HEPES [39.6mL 5%DMEM + 400μL of .22μm filtered 1M HEPES] 7)12 hours or so later (at convenience), take 8 mL of media, filter through .45μm filter and store in 4°C ice bucket. Add 8-8.25mL of DMEM + 5% FBS + 10mM HEPES [39.6mL 5%DMEM + 400μL of .22μm filtered 1M HEPES]
DAY 5
DMEM+5% FBS
1M HEPES
1)12 hours or so later, Aspirate 8mL of media, filter through .45μm filter and store in 4°C ice bucket. Add 8-8.25mL of DMEM + 5% FBS + 10mM HEPES [39.6mL 5%DMEM + 400μL of .22μm filtered 1M HEPES] 2)repeat 12 hours or so later (at convenience) 3)Expose ultracentrifuge tubes and holders to UV for ~1 hour.
DAY 6
TRANSDUCTION
DMEM+5% FBS
1M HEPES
17mL Ultracentrifuge tubes Cells to be infected, conditioned media and fresh media (RAMOS and RPMI + 7% FBS) 8mg/mL polybrene 6-well plate
1)12 hours or so later, Aspirate 8mL of media, filter through .45μm filter and store in 4°C ice bucket. Add 8-8.25mL of DMEM + 5% FBS + 10mM HEPES [39.6mL 5%DMEM + 400μL of .22μm filtered 1M HEPES] 2)Fill 17mL ultracentrifuge tube with 16mL of the media and spin at 70,000 x g average RCF (23,500 rpm at 4°C) for 2.5-3 hours. Repeat until all media is spun down into a single tube. 2 spins per induced plate (8mL*4 collections/16mL=2 spins). Between spins, aspirate/discard media carefully by tilting the tube and not touching the bottom of the tube with the Pasteur pipette. 3)Resuspend in 1mL total of 37°C RPMI, let sit in the hood for 30 minutes to dissolve the virus pellet. 4)Add 5μL of 8mg/mL polybrene to 1,000,000 cells in 1mL conditioned media in one well of a 6-well plate. One protocol says that 8μg/mL final concentration of polybrene. We use 20μg/mL final concentration, 2.5 times that amount. 5)Pipette up and down (no bubbles!) and add the 1mL of virus to transduce
DAY
Conditioned and fresh media for the cells to be infected (in my case RPMI + 7% FBS)
1)After 24 hours, change media. 1 mL new media, 1mL conditioned media.
DAY
1)Check transduction efficiency ~12 hours later
Luciferase activity assay: lysed over 30 minutes, use luciferase assay plates (BioVision luciferase reporter assay kit)
