Constructs and stable cell lines

Lentiviral Production and Transduction

Coat Plates with Poly-L-Lysine

1.  Dilute 20mg/ml poly-L-lysine to 20ug/ml PLL in 1xPBS

50ul PLL(20mg/ml), 5ml 10x PBS, 45 ml ddH2O

2.  Add 4 ml PLL (20ug/ml) to Falcon 10cm plates

3.  Incubate 1 hour at 37°C 

4.  Aspirate PLL solution and wash 3x with 5ml 1xPBS.  To store dishes dry at room temp, rinse 3x with sterile ddH2O and air dry plates in hood.

Passage HEK293T cells from T75 flask

One T75 Flask at 90% Confluency is enough cells to plate three 10cm dishes at 4.5E6 cells.

1.  Aspirate media

2.  Wash with 5ml  2mM EDTA PBS

3.  Add warm 5ml Trypsin-EDTA and gently rock to detach cells

4.  Collect cells in 15ml conical and add 5ml Media to inhibit Trypsin

5.  Spin 300xg , 5 minutes

6.  Aspirate media and flick pellet to loosen cells, resuspend in 10ml media and count cells.

7.  Plate 4.5E6 cells per 10cm dish, cells should quickly adhere to plate.

Day 1: Transfect cells

The following protocol is for transfecting one plate of 293T cells per virus.

1. Change media on cells with 10 ml regular growth media 1 hour prior to transfection 

2. Warm Opti-MEM (OM) at 37°C.

3. Dilute DNA in OM to a total of 1.5ml

4. Dilute 60ul of Lipofectamine 2000 (LF 2000) with 1.440ml OM, let stand at room temperature for 5 minutes.  Solution will become turbid.

5. Add the 1.5ml of LF 2000/OM to the diluted DNA, and let stand for 20 minutes at room temperature.

6. Carefully add to the 10ml media in the plate 3ml of DNA/LF 2000/OM to cells one drop at a time.

7. Incubate overnight for 12-18 hours.

Day 2: Change media

1. Aspirate media and replace with warm Ultra Culture media 10ml per plate

Day 3: 48 hours post transfection, collect virus

1.  Collect media into 15ml conical

2.  Centrifuge supernatant 5 minutes 500xg, at 4°C.

3.  Filter supernatant through 0.45um non-pyrogenic filter

4.  Store supernatant at 4°C while Centricon Plus-20 unit is prepared. 

5.  Sterilize Centricon Plus-20 unit by spraying it with 70% EtOH

6.  Remove glycerin from the unit by adding 19ml of sterile distilled water to sample filter cup.

7.  Centrifuge Centricon Plus-20 unit 15 minutes 2,590xg.

8.  Discard water from collection tube.

9.  Put retentate cup on top of sample filter cup, invert and spin with retentate cup on bottom 1,000xg, 2 minutes.

10.  Aspirate water from retentate cup and replace cup with collection tube.

11.  Add viral supernatant to sample filter cup, cover with cap.

12.  Centrifuge 30 minutes at 2,590xg at 4°C.  Supernatant should completely pass through filter, without any remaining in filter cup portion.

13.  To collect concentrated virus from sample filter cup chambers: place retentate cup on top of sample filter cup, then invert and centrifuge 1000xg for 2 minutes.

14.  Remove from centrifuge keeping orientation with retentate cup on bottom.  Collect the viscous concentrated virus and store at 4°C

Transduce Cells

*We infect 0.5x10^6 THP-1 cells with approximate moi of 10 (293T transduction units).  This equates to an moi of <1 for THP-1 cells (usually around 30% transduction efficiency).  We aim for this relatively low infectivity to ensure that most cells do not have multiple viral integrations.  

**Our average viral titer is 1.175x10^7 viral particles total from 1 plate of 293T cells.  Although each lab should carry out several viral titrations to determine the level and consistency of their titers, we use this average value for THP-1 cell infections and only run titrations if we find our infection rates drop unexpectedly. 

1.  Calculate viral particles per ml by dividing 1.175x10^7 by the total volume of concentrated virus recovered per plate.

2.  Calculate volume of virus needed to infect at 10 moi by dividing 5.0x10^6 by the concentration of viral particles per ml.

3.  Plate 0.5x10^6 cells per well into 2 wells of a 24 well dish.

4.  Infect cells 0.5-3 hours after seeding.

5.  Infect one well by diluting virus up to 500ul with regular growth media supplemented with 4ug/ml polybrene and 100U/ml penicillin G.

6.  Aspirate media, replace with diluted virus.

7.  After 4 hours add 1.5ml regular growth media with penicillin.

8.  The next day split cells 1 x 10^6 cells onto Valmark dishes.

9.  Select for cells 1 to 2 days after split.

Reagents and Materials

293T Growth Media:  DMEM, Gibco # 11995-065 supplemented with 10%FBS and 2mM glutamine

293T falcon dishes 100 x 15 mm TC treated petri dishes, Falcon #353003

Poly-L-Lysine Hydrobromide, Sigma # P1274

Penecillin G, Sigma # P3032

Polybrene, Sigma # H9268

Opti-MEM 1, Gibco # 51985-034

Lipofectamine 2000, Invitrogen #52887

Centricon Plus-20 with 100K NMWL, Millipore #UFC2BHK08

Valmark Ultra-Dish Petri dishes, 100 mm x 15 mm, Midwest Scientific #900 (non-TC treated petri dishes used for passaging of THP-1 cells and BMDMs).  

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