Human CD40 knockout U-2 OS cell line (ab262486)
Overview
-
Product name
Human CD40 knockout U-2 OS cell line -
Parental Cell Line
U-2 OS -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 2 bp insertion; Frameshift: 99.09% -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WB, ICCmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type U-2 OS cell line (ab263976). 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: McCoY5a + 10% FBS
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 2x104 cells/cm2. 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.Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- 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, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
Properties
-
Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Bone -
Cell type
epithelial -
Disease
Osteosarcoma -
Gender
Female -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
-
Function
Receptor for TNFSF5/CD40LG. -
Tissue specificity
B-cells and in primary carcinomas. -
Involvement in disease
Defects in CD40 are the cause of hyper-IgM immunodeficiency syndrome type 3 (HIGM3) [MIM:606843]; also known as hyper-IgM syndrome 3. HIGM3 is an autosomal recessive disorder which includes an inability of B cells to undergo isotype switching, one of the final differentiation steps in the humoral immune system, an inability to mount an antibody-specific immune response, and a lack of germinal center formation. -
Sequence similarities
Contains 4 TNFR-Cys repeats. -
Cellular localization
Secreted and Cell membrane. - Information by UniProt
Associated products
-
KO cell lysates
-
Related Products
- Anti-CD40 antibody (ab113701)
- Anti-CD40 antibody [EPR20540] (ab213205)
- Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free (ab223546)
- Anti-CD40 antibody [EPR20735] (ab224639)
- Anti-CD40 antibody [EPR20735] - BSA and Azide free (ab228818)
- Anti-FHL2 antibody [EPR17860-20] - BSA and Azide free (ab251378)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab262486 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| WB |
Use at an assay dependent concentration.
|
|
| ICC |
Use at an assay dependent concentration.
|
| Notes |
|---|
|
WB
Use at an assay dependent concentration. |
|
ICC
Use at an assay dependent concentration. |
Images
-
Western blot - Human CD40 knockout U-2 OS cell line (ab262486)All lanes : Anti-CD40 antibody [41/CD40] (ab280207) at 1/1000 dilution
Lane 1 : Wild-type U-2 OS Vehicle Control LPS (0µg/mL, 6h) cell lysate
Lane 2 : Wild-type U-2 OS Treated LPS (1µg/mL, 6h) cell lysate
Lane 3 : CD40 knockout U-2 OS Vehicle Control LPS (0µg/mL, 6h) cell lysate
Lane 4 : CD40 knockout U-2 OS Treated LPS (1µg/mL, 6h) cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 4: Merged signal (red and green). Green - ab280207 observed at 45 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55 kDa.
ab280207 was shown to react with CD40 in wild-type U-2 OS cells in Western blot with loss of signal observed in CD40 knockout cell line ab262486 (CD40 knockout cell lysate ab263923). Wild-type U-2 OS and CD40 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab280207 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
-
Next Generation Sequencing - Human CD40 knockout U-2 OS cell line (ab262486)Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 2 bp insertion; Frameshift: 99.09%
-
Western blot - Human CD40 knockout U-2 OS cell line (ab262486)All lanes : Anti-CD40 antibody (ab113701) at 1 µg/ml
Lane 1 : Wild-type U-2 OS cell lysate
Lane 2 : CD40 knockout U-2 OS cell lysate
Lane 3 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab113701 observed at 45 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab113701 was shown to react with CD40 in wild-type U-2 OS cells in Western blot Loss of signal was observed when CD40 knockout cell line ab262486 (knockout cell lysate ab263923) was used. Wild-type and CD40 knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab113701 and 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
-
Immunocytochemistry/ Immunofluorescence - Human CD40 knockout U-2 OS cell line (ab262486)ab228818 staining CD40 in wild-type U-2 OS cells (top panel) and CD40 knockout U-2 OS cells (ab262486) (bottom panel). The cells were fixed with PFA (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab228818 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
-
Western blot - Human CD40 knockout U-2 OS cell line (ab262486)All lanes : Anti-CD40 antibody [EPR20540] (ab213205) at 1/2000 dilution
Lane 1 : Wild-type U-2 OS whole cell lysate
Lane 2 : CD40 knockout U-2 OS whole cell lysate
Lane 3 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab213205 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab213205 was shown to react with CD40 in U-2 OS wild-type cells in Western blot Loss of signal was observed when CD40 knockout cell line ab262486 (knockout cell lysate ab263923) was used. Wild-type U-2 OS and CD40 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab213205 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed 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 hour at room temperature before imaging.
-
Immunocytochemistry/ Immunofluorescence - Human CD40 knockout U-2 OS cell line (ab262486)ab224639 staining CD40 in wild-type U-2 OS cells (top panel) and CD40 knockout U-2 OS cells (ab262486) (bottom panel). The cells were fixed with PFA (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab224639 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Protocols
Datasheets and documents
-
SDS download
-
Datasheet download
References (0)
ab262486 has not yet been referenced specifically in any publications.