Product nameHuman IRAK3 knockout THP-1 cell line
Parental Cell LineTHP-1
Mutation descriptionKnockout achieved by using CRISPR/Cas9, Homozygous: 1 bp + 1 bp deletion in exon 1
Knockout validationSanger Sequencing
Tested applicationsSuitable for: WBmore details
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.
- 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.
Number of cells1 x 106 cells/vial, 1 mL
Cell typeacute monocytic leukemia
DiseaseAcute Monocytic Leukemia
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab281629 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use at an assay dependent concentration.
All lanes : Anti-IRAKM antibody [Y278] (ab32394) at 1/20000 dilution
Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : IRAK3 knockout THP-1 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 68 kDa why is the actual band size different from the predicted?
False colour image of Western blot: Anti-IRAKM antibody [Y278] staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32394 was shown to bind specifically to IRAKM. A band was observed at 68 kDa in wild-type THP-1 cell lysates with no signal observed at this size in IRAK3 knockout cell line ab281629 (knockout cell lysate ab282979). To generate this image, wild-type and IRAK3 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
1 bp + 1 bp deletion in exon 1
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab281629 has not yet been referenced specifically in any publications.