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AB267054

Human CD274 (PD-L1) knockout A549 cell line

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(1 Publication)

CD274 KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. 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. To order both knockout and wild-type control cells: select 2 x 1000000Cells/vial. To order only knockout cells: select 1000000Cells/vial.

View 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

10 Images
Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267054)
  • WB

Lab

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] (ab52866) loading control staining at 1/20000 dilution shown in red. In Western blot 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 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 (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

Lanes 1 - 2:

Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-mouse-igg1-chimeric-ab279292'>ab279292</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-PD-L1 antibody [CAL10] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-ab237726'>ab237726</a>) 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)

Predicted band size: 33 kDa

Observed band size: 48 kDa

false

Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267054)
  • WB

Lab

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

All lanes:

Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-mouse-igg2a-chimeric-ab279293'>ab279293</a>) 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

Observed band size: 45-65 kDa

false

Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267054)
  • WB

Lab

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

Western blot : Anti-CD274 antibody [73-10] (ab228415) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, 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 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] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-73-10-ab228415'>ab228415</a>) 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

false

Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267054)
  • WB

Lab

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

Lanes 1- 6 : Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

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 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. ab213524 and 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 (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.

Lane 1:

Wild-type A549 treated with 100 ng/ml IFN gamma (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 48 h cell lysate at 20 µg

Lanes 2 and 6:

CD274 knockout A549 treated with 100 ng/ml IFN gamma (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) 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

Predicted band size: 33 kDa

Observed band size: 50 kDa

false

Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267054)
  • WB

Lab

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] (ab52866) loading control staining at 1/20000 dilution shown in red. In Western blot 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 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 (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-PD-L1 antibody [CAL10] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-rat-igg2a-chimeric-ab279294'>ab279294</a>) 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)

Predicted band size: 33 kDa

Observed band size: 48 kDa

false

Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (AB267054)
  • Sanger seq

Unknown

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)
  • Sanger seq

Unknown

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)
  • Sanger seq

Unknown

Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (AB267054)

Allele-3 : 1 bp insertion in exon 4.

Cell Culture - Human CD274 (PD-L1) knockout A549 cell line (AB267054)
  • Cell Culture

Unknown

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)
  • Sanger seq

Lab

Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (AB267054)

Sequencing chromatogram displaying sequence edit in exon 4

Key facts

Cell type

A549

Species or organism

Human

Tissue

Lung

Form

Liquid

form

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

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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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.

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What's included?

{ "values": { "2x1000000Cellsvial": { "sellingSize": "2 x 1000000 Cells/vial", "publicAssetCode":"ab267054-2x1000000Cells_vial", "assetComponentDetails": [ { "size":"1 x 1000000 Cells/vial", "name":"ab267054 Human CD274 (PD-L1) knockout A549 cell line", "number":"AB267054-CMP01" }, { "size":"1 x 1000000 Cells/vial", "name":"ab255450 Human wild-type A549 cell line", "number":"AB267054-CMP02" } ] }, "1000000Cellsvial": { "sellingSize": "1000000 Cells/vial", "publicAssetCode":"ab267054-1000000Cells_vial", "assetComponentDetails": [ { "size":"1 x 1000000 Cells/vial", "name":"ab267054 Human CD274 (PD-L1) knockout A549 cell line", "number":"AB267054-CMP01", "productcode":"" } ] } } }
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Properties and storage information

Gene name
CD274
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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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.

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Supplementary information

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

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.

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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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 1 US: 1

Adherent/suspension

Adherent

Gender

Male

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Product protocols

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Target data

See full target information CD274
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Publications (1)

Recent publications for all applications. Explore the full list and refine your search

Oncogenesis 12:33 PubMed37349298

2023

Antibody-based binding domain fused to TCRγ chain facilitates T cell cytotoxicity for potent anti-tumor response.

Applications

Unspecified application

Species

Unspecified reactive species

Zhao Chen,Changyou Lin,Hong Pei,Xiaomei Yuan,Jia Xu,Mingwei Zou,Xinyuan Zhang,Amber Fossier,Meizhu Liu,Seungah Goo,Lei Lei,Jia Yang,Catherine Novick,Jiqing Xu,Ge Ying,Zhihong Zhou,Jianbo Wu,Chunyi Tang,Wenying Zhang,Zhenping Wang,Zhihao Wang,Huitang Zhang,Wenzhong Guo,Qidong Hu,Henry Ji,Runqiang Chen
View all publications
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