Recommended control:Human wild-typeA549 cell line (ab275463). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.

Cryopreservation cell medium:Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

Culture medium:F-12K+ 10% FBS

Initial handling guidelines:Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80ºC. Storage at -80ºC may result in loss of viability.

1. Thaw the vial in 37ºC water bath approximately 1-2 minutes.2. Transfer the cell suspension (0.8 ml) to a 15 ml/50 ml conical sterile polypropylene centrifuge tube containing 8.4 ml pre-warmedculture medium, wash vial with an additional 0.8 mlculture medium(total volume 10 ml) to collect remaining cells, and centrifuge at 201x g(rcf) for 5 minutes at room temperature. 10 ml represents minimum recommended dilution. 20 ml represents maximum recommended dilution.3. Resuspend the cell pellet in 5 ml pre-warmedculture mediumand count using a haemocytometer (Click here to view haemocytometer protocol) or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x10⁴cells/cm². This should allow for confluency within 48 hours. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.4. Incubate the culture at 37ºC incubator with 5% CO₂. Cultures should be monitored daily.

Subculture guidelines:

• All seeding densities should be based on cell counts gained by established methods.
• A guide seeding density of 2x10⁴cells/cm²is recommended for confluency (80-90% confluence) within 48 hours.
• A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
• Cells should be passaged when they have achieved 80-90% confluence.

Click here to view the Mammalian cell tissue culture protocol

This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.