Human CD276 knockout HEK-293T cell line
- Advanced Validation
- What is this?
5
(2 Reviews)
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(0 Publication)
- CD276 KO validation: Immunocytochemistry (ICC),Sanger Sequencing, Western blot
- Concentration: 1 million cells/vial
- WB
Lab
Western blot - Human CD276 knockout HEK-293T cell line (AB266658)
Lanes 1-4 : Merged signal (red and green). Green - ab219648 observed at 90-110 kDa. Red - loading control ab8245 observed at 36 kDa.
ab219648 Anti-CD276 antibody [EPR20115] was shown to specifically react with CD276 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266658 (knockout cell lysate ab257097) was used. Wild-type and CD276 knockout samples were subjected to SDS-PAGE. ab219648 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-CD276 antibody [EPR20115] (<a href='/en-us/products/primary-antibodies/cd276-antibody-epr20115-ab219648'>ab219648</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK293T cell lysate at 20 µg
Lane 2:
CD276 knockout HEK293T cell lysate at 20 µg
Lane 2:
Western blot - Human CD276 knockout HEK-293T cell line (ab266658)
Lane 3:
LNCaP cell lysate at 20 µg
Lane 4:
Raji cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution
Predicted band size: 57 kDa
Observed band size: 90-110 kDa
false
- ICC/IF
Unknown
Immunocytochemistry/ Immunofluorescence - Human CD276 knockout HEK-293T cell line (AB266658)
ab134161 staining CD276 in wild-type HEK293 cells (top panel) and CD276 knockout HEK293 cells (ab266658) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab134161 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha 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 red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
- Flow Cyt
Unknown
Flow Cytometry - Human CD276 knockout HEK-293T cell line (AB266658)
Flow cytometry overlay histogram showing wild-type HEK293 (green line) and CD276 knockout HEK293 cells (ab266658) stained with ab89133 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab89133) (1x106 in 100μl at 0.2 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was mouse IgG1κ (ab170190) used at the same concentration and conditions as the primary antibody (wild-type HEK293 - black line CD276 HEK293 knockout - 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 Cyt
Unknown
Flow Cytometry - Human CD276 knockout HEK-293T cell line (AB266658)
Flow cytometry overlay histogram showing wild-type HEK293 (green line) and CD276 knockout HEK293 cells (ab266658) stained with ab134161 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab134161) (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 HEK293 - black line CD276 HEK293 knockout - 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.
- WB
Lab
Western blot - Human CD276 knockout HEK-293T cell line (AB266658)
Blocking and diluting buffer and concentration : 5% NFDM/TBST.
Performed under reducing conditions.
In Western blot, ab227679 was shown to bind specifically to CD276. Target of interest was observed at 90-100 kDa in wild-type HEK-293T cell lysates (lane 1) with no signal observed at this size in CD276 knockout cell line (lane 2) (lane 2, knockout cell line ab266658.
All lanes:
Western blot - Anti-CD276 antibody [SP265] - C-terminal (<a href='/en-us/products/primary-antibodies/cd276-antibody-sp265-c-terminal-ab227679'>ab227679</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 50 µg
Lane 2:
CD276 knockout HEK-293T whole cell lysate at 50 µg
Lane 2:
Western blot - Human CD276 knockout HEK-293T cell line (ab266658) at 50 µg
Secondary
All lanes:
Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution
Observed band size: 90-110 kDa,36 kDa
false
Exposure time: 26s
- WB
Lab
Western blot - Human CD276 knockout HEK-293T cell line (AB266658)
Lanes 1-4 : Merged signal (red and green). Green - ab227670 observed at 90-110 kDa. Red - loading control ab8245 observed at 36 kDa.
ab227670 Anti-CD276 antibody [SP206] was shown to specifically react with CD276 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266658 (knockout cell lysate ab257097) was used. Wild-type and CD276 knockout samples were subjected to SDS-PAGE. ab227670 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Lanes 1 - 4:
Western blot - Anti-CD276 antibody [SP206] (<a href='/en-us/products/primary-antibodies/cd276-antibody-sp206-ab227670'>ab227670</a>) at 1/1000 dilution
Lanes 1 - 4:
Western blot - Anti-CD276 antibody [SP206], prediluted (<a href='/en-us/products/primary-antibodies/cd276-antibody-sp206-prediluted-ab228178'>ab228178</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK293T cell lysate at 20 µg
Lane 2:
CD276 knockout HEK293T cell lysate at 20 µg
Lane 2:
Western blot - Human CD276 knockout HEK-293T cell line (ab266658)
Lane 3:
LNCaP cell lysate at 20 µg
Lane 4:
Raji cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution
Predicted band size: 57 kDa
Observed band size: 90-110 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human CD276 knockout HEK-293T cell line (AB266658)
Homozygous : Insertion of the selection cassette in exon 2
- Cell Culture
Unknown
Cell Culture - Human CD276 knockout HEK-293T cell line (AB266658)
Representative images of CD276 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.
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Reactivity data
Product details
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. 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.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
DMEM (High Glucose) + 10% FBS
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
CD276 modulates immune responses by acting as a co-inhibitory ligand impacting T-cell proliferation and cytokine synthesis. Not part of a known complex it plays a role in the adaptive immune system by providing negative feedback regulation that can inhibit the immune response. This capability has implications in preventing autoimmunity but may also contribute to tumor immune evasion by reducing effective anti-tumor responses. The B7-H3/CD276 interaction with receptors such as IL receptor family members suggests its diverse immunomodulatory functions.
Pathways
CD276 participates in the T-cell receptor (TCR) signaling pathways influencing immune checkpoint pathways. It interacts with other immune checkpoint proteins like PD-L1 and CTLA-4 but its exact receptor partner remains unidentified. The presence of CD276 in these pathways highlights its role in controlling T-cell activation and tolerance. By modulating TCR signals and impacting downstream effects CD276 serves as a regulatory point that can balance immune activation and inhibition critical in immune-mediated conditions.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com