Mouse CDKN1B (p27 KIP 1) knockout RAW 264.7 cell line

RAW 264.7

Liquid

Knockout achieved by using CRISPR/Cas9,
Homozygous: 52 bp deletion-3 bp insertion in Exon 1

Carcinoma

CDKN1B

Knockout

CRISPR technology

Sanger Sequencing, Western blot

Homozygous

EU: 2 US: 2

Adherent

Male

Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

1. Thaw the vial in 37°C water for 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-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x 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-warmed culture medium and count using a haemocytometer or alternative cell counting method all remaining cells into an ultra-low attachment T25.
4. Incubate the culture at 37°C incubator with 5% CO₂. Check the culture one day after revival and continue to check until 80% confluent.
5. Once confluent passage into an appropriate flask at a density of 2x10⁵ cells/cm². Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.

• Grow in ultra-low attachment flasks.


• Do not wash or use dissociation reagent. Cells are semi-adherent. Tap flask sharply to remove adhered cells.


• All seeding densities should be based on cell counts gained by established methods.


• A guide seeding density of 2x10⁵ cells/mL is recommended.


• Cells should be passaged when they have achieved 80-90% confluence.

DMEM + 10% FBS

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