ViroVision HRC Culture Protocol
1. Results are only guaranteed if ViroVision™ Infection Enhancement Medium is used when cells are infected with HIV.
2. Once thawed, the Infection Enhancement Medium should be stored at 4°C, and is stable for 3 months. Do not re-freeze and do not leave Infection Medium at room temperature.
3. Depending on the cell line, there may be different media requirements. Please consult the table at the end to match proper media with purchased cell line.
Intitiation of ViroVision™ Cell Culture
- Please ensure cells arrive frozen. Contact Cube Biosystems immediately if cells arrive thawed.
- Cells should be stored in liquid nitrogen if not to be used immediately.
- To initiate cell culture, remove a vial of frozen cells from storage, and thaw as quickly as possible by placing in a 37°C water bath.
Note: If cells are removed from liquid nitrogen, under sterile conditions, unscrew cap ½ to 1 full turn to allow N2 gas to escape. Re-secure cap and place cells in 37°C water bath to thaw.
- Once thawed, transfer the cell suspension to a sterile 15 ml tube. Add 5 ml complete media dropwise, mix gently after each addition.
- Collect the cells by centrifugation at 300 x g for 5 minutes, room temperature.
- Remove/aspirate the supernatant and resuspend the cell pellet in 15 ml of complete media.
- Place the cells into a T75 flask and incubate at 37°C, 5% CO2.
Culturing ViroVision™ Cells
- Count cells daily and keep at a density below 1 x 10^6 cells/ml. Dead cells may be removed by Ficoll separation.
- Add fresh medium (RPMI complete) when cell density reaches 1 x 10^6 per ml.
- Count cells and pellet cells by centrifugation at 300 x g for 5 minutes.
Note: Cell viability should be ≥80%.
- Resuspend cells in complete media at concentration of ~ 2 x 10^6 cells/ml.
- Use 100 µl of cells (~2 x 10^5) per infection.
- Pre-treat cells by adding 10 µl of ViroVision™ Infection Enhancement Media (10X) so that Infection Media concentration is ~ 1X. Mix and incubate for 2 hours. Use of ViroVision™ Infection Enhancement Medium is required.
- Add virus to the cells & mix. Note volume of virus used.
- Add Infection Enhancement Media to 1/10 of the virus volume used. E.g. If 100 µl of virus used, add 10 µl of Infection Medium. Incubate at 37°C for 2 - 4 hours.
- Add 1 ml fresh complete media to wash cells. Pellet cells as above and remove supernatant. (Optional) Repeat 1X for a total of 2 washes.
- After washing cells, resuspend cells in 1 ml RPMI complete medium.
- Culture cells at 37°C, 5% CO2 for 2-5 days. GFP or Luciferase may be quantified 48 hours after infection.
ViroVision™ Media Guide
|Rev-A3R5||ViroVision™ Growth Media A:
RPMI-1640 containing 10-15% FBS, 1% L-Glut, 1% Pen/Strep, and 1 mg/mL Geneticin (G418)
|Rev-A3||ViroVision™ Growth Media B:
RPMI-1640 containing 10-15% FBS, 1% L-Glut, 1% Pen/Strep
|Rev-CEM||ViroVision™ Growth Media C:
RPMI, 10% FBS