Human CD274 (PD-L1) knockout A549 cell line (ab267054)
Overview
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Product name
Human CD274 (PD-L1) knockout A549 cell line
See all PD-L1 lysates -
Parental Cell Line
A549 -
Organism
Human -
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 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General 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.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: F-12K + 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 2x103-1x104 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 6x104 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.
- Do not exceed 7x104 cells/cm2.
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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Lung -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
STR Analysis
Amelogenin X,Y D5S818: 11 D13S317: 11 D7S820: 8, 11 D16S539: 11, 12 vWA: 14 TH01: 8,9.3 TPOX: 8,11 CSF1PO: 10, 12 -
Antibiotic resistance
Puromycin 1.00µg/ml -
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
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Function
Involved in the costimulatory signal, essential for T-cell proliferation and production of IL10 and IFNG, in an IL2-dependent and a PDCD1-independent manner. Interaction with PDCD1 inhibits T-cell proliferation and cytokine production. -
Tissue specificity
Highly expressed in the heart, skeletal muscle, placenta and lung. Weakly expressed in the thymus, spleen, kidney and liver. Expressed on activated T- and B-cells, dendritic cells, keratinocytes and monocytes. -
Sequence similarities
Belongs to the immunoglobulin superfamily. BTN/MOG family.
Contains 1 Ig-like C2-type (immunoglobulin-like) domain.
Contains 1 Ig-like V-type (immunoglobulin-like) domain. -
Cellular localization
Cell membrane and Endomembrane system. - Information by UniProt
Associated products
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KO cell lysates
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab267054 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. Predicted molecular weight: 33 kDa.
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| Notes |
|---|
|
WB
Use at an assay dependent concentration. Predicted molecular weight: 33 kDa. |
Images
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Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)All lanes : 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
Lane 2 : Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate
Lane 3 : CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) cell lysate
Lane 4 : CD274 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 48 h) cell lysate
Lane 5 : Human Placenta cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 45-65 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Mouse IgG2a staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279293 was shown to bind specifically to PD-L1. A band was observed at 45-65 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 fluorescent western blot (TBS-based) blocking solution 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.
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Sanger Sequencing - Human CD274 (PD-L1) knockout A549 cell line (ab267054)Sequencing chromatogram displaying sequence edit in exon 4 -
Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)All lanes : 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
Lane 2 : CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-PD-L1 antibody [CAL10] - Rat IgG2a 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 °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-Rat IgG H&L (IRDye® 800CW) preabsorbed (ab253031) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)All lanes : Anti-PD-L1 antibody [CAL10] – Mouse IgG1 (Chimeric) (ab279292) at 1/1000 dilution
Lane 1 : Wild-type A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate
Lane 2 : CD274 knockout A549 Treated IFN-gamma (100 ng/ml) for 48 hours cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?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 °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 (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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Western blot - Human CD274 (PD-L1) knockout A549 cell line (ab267054)All lanes : Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution
Lane 1 : Wild-type A549 treated with 100 ng/ml IFN gamma (ab259377) for 48 h cell lysate
Lane 2 : CD274 knockout A549 treated with 100 ng/ml IFN gamma (ab259377) for 48 h cell lysate
Lane 3 : U-87 MG cell lysate
Lane 4 : MCF7 cell lysate
Lane 5 : Wild-type A549 untreated cell lysate
Lane 6 : CD274 knockout A549 untreated cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 33 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?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°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.
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Sanger Sequencing - Human CD274 knockout A549 cell line (ab267054)Allele-1: 7 bp deletion in exon4
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Sanger Sequencing - Human CD274 knockout A549 cell line (ab267054)Allele-2: 2 bp deletion in exon 4.
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Sanger Sequencing - Human CD274 knockout A549 cell line (ab267054)Allele-3: 1 bp insertion in exon 4.
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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.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab267054 has not yet been referenced specifically in any publications.