Human IRAK3 knockout THP-1 cell line (ab281629)

Recommended control: Human wild-type THP-1 cell line (ab271147). 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: RPMI + 10% FBS + 0.05 mM β-mercaptoethanol

Initial handling guidelines: 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 bath for 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. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2-4x10⁵ cells/mL. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO₂. Cultures should be monitored daily.
5. THP-1 cells recover slowly from cryopreservation and therefore may not be ready for subculture for a number of days. Cells should be left as much as possible over this time and only subcultured when the cell density reaches 8x10⁵ cells/mL.

Subculture guidelines:

• All seeding densities should be based on cell counts gained by established methods.
• Cells should be seeded at 2-4x10⁵ cells/mL and subcultured when they have reached 8x10⁵ cells/mL. It is not recommended to allow the cell density to exceed 1x10⁶ cells/mL.
• 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.

We will provide viable cells that proliferate on revival.