Human CD274 (PD-L1) knockout A549 cell line (ab267054)

CD274 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 4 and 2 bp deletion in exon 4 and 7 bp deletion in exon 4.

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Images

Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267054), expandable thumbnail

Publications

Key facts

Cell type: A549
Species or organism: Human
Tissue: Lung
Form: Liquid
Knockout validation: Sanger Sequencing, Western blot
Mutation description: Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 4 and 2 bp deletion in exon 4 and 7 bp deletion in exon 4

Alternative names

B7 H, B7 homolog 1, B7-H1, CD-274, CD274 antigen, CD274 molecule, MGC142294, MGC142296, OTTHUMP00000021029, PD-L1, PD1L1_HUMAN, PDCD1 ligand 1, PDCD1L1, PDCD1LG1, Programmed cell death 1 ligand 1, Programmed death ligand 1, RGD1566211

Key facts

Cell type: A549
Species or organism: Human
Tissue: Lung
Form: Liquid
Knockout validation: Sanger Sequencing, Western blot
Mutation description: Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 4 and 2 bp deletion in exon 4 and 7 bp deletion in exon 4
Antibiotic resistance: Puromycin 1µg/mL
Disease: Carcinoma
Concentration

Properties

Gene name: CD274
Gene editing type: Knockout
Gene editing method: CRISPR technology
Knockout validation: Sanger Sequencing, Western blot

Quality control

STR analysis: CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

Cell culture

Biosafety level: EU: 1 US: 1
Adherent/suspension: Adherent
Gender: Male

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.
Do not allow the cell density to exceed 7x10⁴ cells/cm².

Culture medium

F-12K + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

Storage

Shipped at conditions: Dry Ice
Appropriate short-term storage conditions: -196°C
Appropriate long-term storage conditions: -196°C

Notes

Recommended control: Human wild-type A549 cell line (ab255450). 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, 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.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

PD-L1 also known as Programmed Death-Ligand 1 or CD274 is a protein involved in immune modulation. Mechanically PD-L1 interacts with its receptors particularly PD-1 to regulate cellular immune responses. This transmembrane protein has a calculated molecular weight of approximately 33 kDa. PD-L1 is expressed on various cell types including tumor cells and immune cells such as dendritic cells macrophages and B cells. Its expression is often upregulated in response to inflammatory cytokines.

Biological function summary

PD-L1 plays a central role in immune evasion mechanisms utilized by tumors. It is not part of a larger protein complex but directly interacts with PD-1 and CD80. When PD-L1 binds to PD-1 it sends inhibitory signals leading to decreased T cell activation and proliferation allowing cancer cells to avoid immune destruction. PD-L1 expression provides a mechanism for tumors to suppress immune surveillance facilitating tumor progression.

Pathways

PD-L1 is integral to the immune checkpoint pathway which is an important regulator of immune response. The interaction between PD-L1 and PD-1 provides a mechanism for immune tolerance which is part of the broader adaptive immune system pathway. PD-L1 is related to other immune checkpoint proteins such as CTLA-4 in its function to limit autoreactivity and promote immune homeostasis.

Associated diseases and disorders

PD-L1 is most associated with cancer particularly in tumors such as melanoma and non-small cell lung cancer. PD-L1 expression on tumor cells often correlates with poor prognosis. PD-L1 directly interacts with PD-1 in these cancers a target for immunotherapies such as checkpoint inhibitors which aim to block this interaction and restore immune activity against tumors. PD-L1 involvement extends to autoimmune disorders where altered expression can impact tolerance and lead to immune-related tissue damage.

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10 product images

Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
False colour image of Western blot: Anti-PD-L1 antibody [CAL10] – Rat IgG2a (Chimeric) staining at 1/1000 dilution shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) ab279294 was shown to bind specifically to PD-L1. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in Cd274 knockout cell line ab267054 (knockout cell lysate Human CD274 (PD-L1) knockout A549 cell lysate ab256831). To generate this image wild-type and Cd274 knockout A549 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°. 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-Rat IgG H&L (IRDye^® 800CW) preabsorbed (ab253031) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
All lanes: Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) ab279294) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Lane 2: CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Lane 2: Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 48 kDa
Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Lanes 1- 6: Merged signal (red and green). Green - Anti-PD-L1 antibody [EPR19759] ab213524 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
Anti-PD-L1 antibody [EPR19759] ab213524 was shown to react with PD-L1 in wild-type A549 treated with 100 ng/ml IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell lines ab267054 ( treated and untreated knockout cell lysates Human CD274 (PD-L1) knockout A549 cell lysate ab256831) were used. Wild-type A549 treated with 100 ng/ml IFN gamma for 48 h and CD274 knockout A549 treated with 100 ng/ml IFN gamma for 48 h cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-PD-L1 antibody [EPR19759] ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4° at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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.
Lane 1: Wild-type A549 treated with 100 ng/ml IFN gamma (Recombinant Human Interferon gamma protein (Active) ab259377) for 48 h cell lysate at 20 µg
Lanes 2 and 6: CD274 knockout A549 treated with 100 ng/ml IFN gamma (Recombinant Human Interferon gamma protein (Active) ab259377) for 48 h cell lysate at 20 µg
Lane 2: Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Lane 3: U-87 MG cell lysate at 20 µg
Lane 4: MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 untreated cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 50 kDa
Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
False colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Mouse IgG1 staining at 1/1000 dilution shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) ab279292 was shown to bind specifically to PD-L1. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in Cd274 knockout cell line ab267054 (knockout cell lysate Human CD274 (PD-L1) knockout A549 cell lysate ab256831). To generate this image wild-type and Cd274 knockout A549 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 °. 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-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) at 1/20000 dilution.
Lanes 1 - 2: Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) (Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) ab279292) at 1/1000 dilution
Lanes 1 - 2: Western blot - Anti-PD-L1 antibody [CAL10] (Anti-PD-L1 antibody [CAL10] ab237726) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Lane 2: CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate at 20 µg
Lane 2: Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 48 kDa
Cell Culture - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Representative images of CD274 knockout A549 cells, low and high confluency examples (top left and right respectively) and wild-type A549 cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Allele-3: 1 bp insertion in exon 4.
Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Western blot: Anti-CD274 antibody [73-10] (Anti-PD-L1 antibody [73-10] ab228415) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-PD-L1 antibody [73-10] ab228415 was shown to bind specifically to CD274. A band was observed at 40-60 kDa in treated wild-type A549 cell lysates with no signal observed at this size in CD274 knockout cell line ab267054 (knockout cell lysate Human CD274 (PD-L1) knockout A549 cell lysate ab256831). To generate this image, wild-type and CD274 knockout A549 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-PD-L1 antibody [73-10] (Anti-PD-L1 antibody [73-10] ab228415) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
Lane 3: CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 4: CD274 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
Secondary
All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
All lanes: Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) (Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) ab279293) at 1/1000 dilution
Lane 1: Wild-type A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
Lane 3: CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate at 20 µg
Lane 4: CD274 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate at 20 µg
Lane 5: Human Placenta cell lysate at 20 µg
Secondary
Lanes 1 - 5: Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 5: Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 45-65 kDa
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Allele-2: 2 bp deletion in exon 4.
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Allele-1: 7 bp deletion in exon4
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Sequencing chromatogram displaying sequence edit in exon 4