CD74 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 13 bp deletion in exon 2.
CD74 antigen, CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated), CD74 molecule, CD74 molecule, major histocompatibility complex, class II invariant chain, DHLAG, Gamma chain of class II antigens, HG2A_HUMAN, HLA class II histocompatibility antigen gamma chain, HLA-DR antigens-associated invariant chain, HLA-DR-gamma, HLADG, Ia antigen-associated invariant chain, Ia associated invariant chain, Ia gamma, Ii, Invariant polypeptide of major histocompatibility complex class II antigen associated, MHC HLA-DR gamma chain, Major histocompatibility complex class II invariant chain, P35, Protein 41, la-gamma, p33
CD74 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 13 bp deletion in exon 2.
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. Based on cell count, seed cells in an appropriate cell culture flask at a density of 4x105 - 5x105 cells/mL(for initial passages it is recomended to culture the cells in the higher range of recomended seeding density). 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.
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
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.
We will provide viable cells that proliferate on revival.
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.
CD74 also known as the invariant chain or li is a protein with a mass of about 33 kDa. It is widely expressed on the surface of immune cells such as B cells dendritic cells and macrophages. CD74 plays an important role in the immune system by acting as a chaperone for major histocompatibility complex class II (MHC II) molecules guiding their migration to endosomes where they encounter antigenic peptides. The CD74 protein also functions as a cell surface receptor for macrophage migration inhibitory factor (MIF) enhancing the immune response process.
CD74 facilitates multiple immune regulatory processes. It forms a complex with the MHC II molecules aiding in their proper folding and peptide loading in the endoplasmic reticulum. This complex then travels through the Golgi apparatus towards the lysosomal compartments where antigens are presented to initiate adaptive immune responses. In addition CD74 is involved in signal transduction pathways that regulate cell proliferation and survival influenced by interactions with MIF and other molecules.
The role of CD74 extends into both the antigen presentation and MIF signaling pathways. Within the antigen presentation pathway CD74's activity is tightly connected with MHC II and subsequently to CD4+ T cells facilitating the activation of these immune cells. The MIF signaling pathway involves CD74 interacting with CD44 a receptor that further propagates the signaling cascades essential for immune modulation and cell survival.
CD74 is linked to inflammatory and autoimmune conditions such as rheumatoid arthritis and systemic lupus erythematosus. Aberrant expression of CD74 influences the presentation of autoantigens and may contribute to the chronic inflammation seen in these disorders. Its connection to MIF which is a pro-inflammatory cytokine further implicates CD74 in these autoimmune scenarios by promoting persistent immune activation. Researchers are also exploring CD74 as a therapeutic target in certain cancers due to its influence on immune surveillance and tumor progression.
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Flow cytometry overlay histogram showing wild-type Raji (green line) and CD74 knockout Raji cells (ab273378) stained with Anti-CD74 antibody [CerCLIP.1] ab22606 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (Anti-CD74 antibody [CerCLIP.1] ab22606) (1x106 in 100μl at 5 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor® 488, pre-adsorbed) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was mouse IgG1κ (Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control ab170190) used at the same concentration and conditions as the primary antibody (wild-type Raji cells - black line; CD74 knockout Raji cells ab273378 - 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.
Flow cytometry overlay histogram showing wild-type Raji (green line) and CD74 knockout Raji cells (ab273378) stained with Anti-CD74 antibody [CLIP/3127R] ab270265 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (Anti-CD74 antibody [CLIP/3127R] ab270265) (1x106 in 100μl at 1 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor® 488, pre-adsorbed) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was Rabbit IgG (monoclonal) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) used at the same concentration and conditions as the primary antibody (wild-type Raji cells - black line; CD74 knockout Raji cells ab273378 - 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.
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD74 antibody [CLIP/3127R] ab270265 observed at 35 kDa. Red - loading control, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
Anti-CD74 antibody [CLIP/3127R] ab270265 was shown to react with CD74 in western blot. The band observed in CD74 CRISPR/Cas9 edited cell line ab273378 (CRISPR/Cas9 edited lysate Human CD74 knockout Raji cell lysate ab275529) below 35 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-CD74 antibody [CLIP/3127R] ab270265 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-CD74 antibody [CLIP/3127R] (Anti-CD74 antibody [CLIP/3127R] ab270265) at 1 µg/mL
Lane 1: Wild-type Raji cell lysate at 30 µg
Lane 2: CD74 knockout Raji cell lysate at 30 µg
Lane 2: Western blot - Human CD74 knockout Raji cell line (ab273378)
Lane 3: Jurkat cell lysate at 30 µg
Lane 4: HepG2 cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 34 kDa
Observed band size: 35 kDa
Anti-CD74 antibody [EPR4064] ab108393 was shown to react with CD74 in western blot. The band observed in CD74 knockout cell line ab273378 (knockout lysate Human CD74 knockout Raji cell lysate ab275529) below 35 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with Anti-CD74 antibody [EPR4064] ab108393 and Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-CD74 antibody [EPR4064] (Anti-CD74 antibody [EPR4064] ab108393) at 1/1000 dilution
Lane 1: Wild-type Raji cell lysate at 30 µg
Lane 2: Western blot - Human CD74 knockout Raji cell lysate (Human CD74 knockout Raji cell lysate ab275529)
Lane 2: CD74 knockout Raji cell lysate at 30 µg
Lane 2: Western blot - Human CD74 knockout Raji cell line (ab273378)
Lane 3: Jurkat cell lysate at 30 µg
Lane 4: HepG2 cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 34 kDa
Observed band size: 35 kDa
Anti-CD74 antibody ab64772 was shown to react with CD74 in western blot. The band observed in CD74 knockout cell line ab273378 (knockout lysate Human CD74 knockout Raji cell lysate ab275529) below 35 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with Anti-CD74 antibody ab64772 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-CD74 antibody (Anti-CD74 antibody ab64772) at 1 µg/mL
Lane 1: Wild-type Raji cell lysate at 30 µg
Lane 2: CD74 knockout Raji cell lysate at 30 µg
Lane 2: Western blot - Human CD74 knockout Raji cell lysate (Human CD74 knockout Raji cell lysate ab275529)
Lane 2: Western blot - Human CD74 knockout Raji cell line (ab273378)
Lane 3: Jurkat cell lysate at 30 µg
Lane 4: HepG2 cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 34 kDa
Observed band size: 35 kDa
Flow cytometry overlay histogram showing wild-type Raji (green line) and CD74 knockout Raji cells (ab273378) stained with Anti-CD74 antibody [EPR4064] ab108393 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (Anti-CD74 antibody [EPR4064] ab108393) (1x106 in 100μl at 0.2 μg/ml) for 30 min at 22°C.
The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor® 488, pre-adsorbed) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was used at 1/2000 for 30 min at 22°C.
Isotype control antibody was Rabbit IgG (monoclonal) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) used at the same concentration and conditions as the primary antibody (wild-type Raji cells - black line; CD74 knockout Raji cells ab273378 - 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.
All lanes: Western blot - Anti-CD74 antibody [PIN.1] (Anti-CD74 antibody [PIN.1] ab22603) at 1 µg/mL
Lane 1: Wild-type Raji cell lysate at 30 µg
Lane 2: Western blot - Human CD74 knockout Raji cell lysate (Human CD74 knockout Raji cell lysate ab275529)
Lane 2: CD74 knockout Raji cell lysate at 30 µg
Lane 2: Western blot - Human CD74 knockout Raji cell line (ab273378)
Lane 3: Jurkat cell lysate at 30 µg
Lane 4: HepG2 cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 34 kDa
Observed band size: 35 kDa
Flow cytometry overlay histogram showing wild-type Raji (green line) and CD74 knockout Raji cells (ab273378) stained with Anti-CD74 antibody [LN2] ab9514 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (Anti-CD74 antibody [LN2] ab9514) (1x106 in 100μl at 1 μg/ml) for 30 min at 22°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor® 488, pre-adsorbed) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was used at 1/2000 for 30 min at 22°C.
Isotype control antibody was mouse IgG1κ (Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control ab170190) used at the same concentration and conditions as the primary antibody (wild-type Raji cells - black line; CD74 knockout Raji cells ab273378 - 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.
This antibody gave a positive signal in CD74 knockout Raji cells fixed with 4% formaldehyde (10 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.
Anti-CD74 antibody [LN2] ab9514 was shown to react with CD74 in western blot. The band observed in CD74 knockout cell line ab273378 (knockout lysate Human CD74 knockout Raji cell lysate ab275529) below 35 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with Anti-CD74 antibody [LN2] ab9514 and Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4 ° at 5 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-CD74 antibody [LN2] (Anti-CD74 antibody [LN2] ab9514) at 5 µg/mL
Lane 1: Wild-type Raji cell lysate at 30 µg
Lane 2: CD74 knockout Raji cell lysate at 30 µg
Lane 2: Western blot - Human CD74 knockout Raji cell lysate (Human CD74 knockout Raji cell lysate ab275529)
Lane 2: Western blot - Human CD74 knockout Raji cell line (ab273378)
Lane 3: Jurkat cell lysate at 30 µg
Lane 4: HepG2 cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 34 kDa
Observed band size: 35 kDa
Homozygous: 13 bp deletion in exon 2
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