Product nameHuman CXCR5 knockout Raji cell line
Parental Cell LineRaji
Mutation descriptionKnockout achieved by using CRISPR/Cas9, Homozygous: 19 bp deletion in exon 2
Knockout validationSanger Sequencing, Western Blot (WB)
Tested applicationsSuitable for: WB, Flow Cytmore details
Recommended control: Human wild-type Raji cell line (ab275473). 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
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-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 (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 2x104 cells/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.
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is 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.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Number of cells1 x 106 cells/vial, 1 mL
Cell typeBurkitt's lymphoma
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
PurityImmunogen affinity purified
FunctionCytokine receptor that binds to B-lymphocyte chemoattractant (BLC). Involved in B-cell migration into B-cell follicles of spleen and Peyer patches but not into those of mesenteric or peripheral lymph nodes. May have a regulatory function in Burkitt lymphoma (BL) lymphomagenesis and/or B-cell differentiation.
Tissue specificityExpression in mature B-cells and Burkitt lymphoma cells.
Sequence similaritiesBelongs to the G-protein coupled receptor 1 family.
Cellular localizationCell membrane.
- Information by UniProt
KO cell lysates
Our Abpromise guarantee covers the use of ab273380 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration. Predicted molecular weight: 42 kDa.|
|Flow Cyt||Use at an assay dependent concentration.|
All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution
Lane 1 : Wild-type Raji cell lysate
Lane 2 : CXCR5 knockout Raji cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : Jurkat cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Predicted band size: 42 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?
ab254415 was shown to react with CXCR5 in Raji wild-type cells in Western blot with loss of signal observed in CXCR5 knockout sample. Wild-type and CXCR5 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab254415 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
Flow cytometry overlay histogram showing wild-type Raji (green line) and CXCR5 knockout Raji cells (ab273380) stained with ab254415 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serumto block FC receptors and non-specific protein-protein interaction followed by the antibody (ab254415) (1x106 in 100μl at 0.2 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type Raji - black line; CXCR5 knockout Raji - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
Homozygous: 19 bp deletion in exon 2
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.
ab273380 has not yet been referenced specifically in any publications.