Human CD274 (PD-L1) knockout A549 cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Lab
Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267055)
False colour image of Western blot : Anti-PD-L1 antibody [CAL10] – Mouse IgG1 (Chimeric); Rabbit anti-Vinculin antibody (ab219649) loading control staining at 1/1000 dilution, shown in red. In Western blot, ab279292 was shown to bind specifically to PD-L1. A band was observed at 45 kDa in wild-type A549 cell lysates with no signal observed at this size in Cd274 knockout cell line. 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
All lanes:
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 - Human wild-type A549 cell line (ab255450)
Lane 1:
Wild-type A549 Control IFN-gamma (0 ng/mL, 48 h) at 20 µg
Lane 2:
Wild-type A549 Treated IFN-gamma (100 ng/mL, 48 h) at 20 µg
Lanes 3 - 4:
Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267055)
Lane 3:
CD274 knockout A549 Control IFN-gamma (0 ng/mL, 48 h) at 20 µg
Lane 4:
CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) at 20 µg
Secondary
All lanes:
Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution
Predicted band size: 33 kDa
Observed band size: 45 kDa
false
- WB
Lab
Western blot - Human CD274 (PD-L1) knockout A549 cell line (AB267055)
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 ab267055 (treated and untreated knockout cell lysates ab256866) 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°C 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.
All lanes:
Western blot - Anti-PD-L1 antibody [EPR19759] (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-epr19759-ab213524'>ab213524</a>) at 1/1000 dilution
Lane 1:
Wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cell lysate at 20 µg
Lane 2:
CD274 knockout A549 treated with 100 ng/mL IFN gamma for 48 h cell lysate at 20 µg
Lane 2:
Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267055)
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
Lane 6:
CD274 knockout A549 untreated cell lysate at 20 µg
Predicted band size: 33 kDa
Observed band size: 50 kDa
false
- Cell Culture
Unknown
Cell Culture - Human CD274 (PD-L1) knockout A549 cell line (AB267055)
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 seq
Unknown
Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (AB267055)
Homozygous : 1 bp insertion in exon4
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Cell Lines & Lysates
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Advanced Validation
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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.
- 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.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
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.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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