- Transduction Day 2: change media
Transduction Day 2
- Brought 25 mL aliquots of media for each cell line to the LaBaer lab.
- Checked GFP control wells (LaBaer scope has no RFP filters)
- Plate 1: U2OS, ~90% GFP expression, at least ~two doubling
- Plate 2: SK-N-SH, ~90% expression, slow cell growth
- Plate 4: K562, no GFP expression. Did not continue next steps for this plate
- Remove media
- Add 2 ml/well fresh media with 1% Pen/strep
- Grow cells at 37°C (overnight)
Technical hint from Research Gate
- In our lab, we concentrate our lentivirus after production using PEG-precipitation. This increases the concentration 50-100x. Then, we "spinoculate" K562 cells with a small amount of virus in an eppendorf tube. Typically, this means putting 50,000 - 100,000 K562 cells in an eppendorf tube, adding 1-2 uL of lentivirus, adding polybrene to 4 ug/ml, and centrifuging for 30 min at 800 x g (at 32 C or room temperature). If your lentivirus is of good quality, this method gives very high infection rate (normally I get greater than 90%...close to 100).
- Mark Duncan, Northwestern U.
Trouble-shooting. Maybe try this
- Put 80k/1 mL K562 cells + Polybrene + 0.4 mL viral aliquot into 15 mL conicals
- Spin, according to Ganzalez protocol (9/23/14)
- Resuspend and plate total volume in 6-well plate