Unit size: 1 million cells per vial
Bio-Safety: Level II
Origin: Human Peripheral Blood
Growth Properties: Suspension
Description: HL-60 cells stably expressing Cas9
Population Doubling: 24-40 hours
Selection drug: Puromycin
1. Pre-warm complete cell culture media in a 37°C waterbath.
2. Remove the cryopreserved vial from the liquid nitrogen storage tank.
3. Thaw the cells quickly at 37°C water bath and remove after 60 seconds. There should still be a few ice crystals left after thawing. It is important not to over-thaw the cryovials as the presence of DMSO is toxic to the cells.
4. Re-suspend the cells in the vial and transfer the cell suspension into a 15mL sterile conical tube containing 5ml complete culture media.
5. Centrifuge cells at 200x g for 10 minutes at room temperature.
6. Aspirate out the media, leaving cell pellet undisturbed.
7. Re-suspend pellet in 10 ml fresh culture medium and plate in new culture vessel T25.
8. Incubate cultures at 37°C, 5% CO2.
Note: Where applicable, we recommend selection drug addition after cells recovered from thawing. Directly adding selection drug while thawing could lead to stressed cells and lower viability.
Propagation and subculture:
The base medium for this cell line is Prigrow V medium available at abm, Cat. No. (ABMTM015). To make the completed growth medium, add the following components to the base medium with the final concentration: 20% fetal bovine serum (ABMTM999), 25mM L-Glutamine, 0.6 µg/ml puromycin (ABMG264), and Penicillin/Streptomycin Solution (ABMG255) to a final concentration of 1%.
1) Puromycin selection
2) Western Blot analysis for Cas9 transgene expression
CO2: 5%, at 37.0°C