Editing Lentivirus Concentration by Ultrafiltration

1. Grow low passage (&lt;12) 293T cells below confluence in 500 cm2 TripleFlasks in 150 mL D10 medium

(DMEM with 10% fetal bovine serum, 50 U/mL penicillin,50 ug/mL streptomycin, and 2 mM l-glutamine).

Passage cells at 1:4–1:5 every 2 days, or 1:8–1:10 every 3 days.

2. Transfect cells with lentivirus plasmids mix

Add 1 mL GeneExpresso to 50 mL of DMEM

Add <br>150 ug of vector plasmid, <br>150 ug of pGagPol <br>30 ug of pCMV-VSVG <br>incubated for 20 min at room temperature.

<br>3. Wash cellwith 15 mL 0.05% trypsin EDTA in HBSS to bind and remove trypsin inhibitors.<br>digest with 15 mL of 0.05% trypsin incubate for 3–5 min incubation at 37 oC.<br>Add 15 mL of D10 to inactivate the trypsin.<br>Transfer cells a sterile polystyrene bottle.<br>Rinse TripleFlask with 30 mL of D10 .<br>Combine cells.

4. Add transfection mix to 7 × 10^8 cells (from three TripleFlasks, NUNC 132867) in ∼150 mL D10. 

5. Transfer cells/transfection mix to a HYPERFlask. Add D10 to dislodge air bubbles from the layers

6. Place the flask in a horizontal position at 37 oC overnight.

7. Change medium 18–20 h post-transfection: D10 containing 10 mM sodium butyrate and 20 mM HEPES.

8. After 6–8 h, rinse cells once with 500 mL DPBS and then add fresh harvesting medium, consisting of UltraCULTURE<br>with 20 mM HEPES, 50 U/mL penicillin, 50 ug/mL streptomycin, and 2 mM l-glutaminewas added to HYPERFlask (Corning 10010.

Use one finger to gently tap the side of the flask, while held in a vertical position to dislodge air without appreciably disrupting the cell monolayers.

9. About 40 h after addition of harvesting medium, harvest LV-containing medium from HYPERFlasks, store medium at 4 oC.

10. Add fresh medium.

11. Harvest medium 24 h later

12. Filter medium with 0.45 or 0.8 micron filter. Combine filtered medium

13. Concentrate filtered medium using KrosFlo Research II TFF System<br>KrosFlo Research II TFF System (Spectrum Labs, SYR2-U20-01 N).<br>Flow path 1 [FPI] (Spectrum Labs, 500 kDa, EZ-M1-500S-260-01N-I).<br>Flow path 2 [FPII] (Spectrum Labs, EZ-CHIL07-01-I).<br><br>

[[Category:Protocols]]