VCAM1 KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 4 bp deletion in exon 2.
Images
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 4 bp deletion in exon 2
Alternative names
CD106, CD106 Antigen, INCAM-100, MGC99561, VCAM1_HUMAN, Vascular Cell Adhesion Molecule 1, Vascular cell adhesion protein 1
Recommended products
VCAM1 KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 4 bp deletion in exon 2.
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 4 bp deletion in exon 2
- Disease
- Carcinoma
- Concentration
Properties
- Gene name
- VCAM1
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Zygosity
- Homozygous
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
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant 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
VCAM1 also known as vascular cell adhesion molecule 1 plays an important role in mediating adhesion and signal transduction. VCAM1 has an approximate molecular mass of 110 kDa. It is expressed on the surface of endothelial cells and can be upregulated by cytokines released during inflammation. The protein serves as a ligand for the integrin VLA-4 also known as α4β1 and contributes to the adhesion of leukocytes to endothelial cells.
- Biological function summary
Adhesion molecules like VCAM1 participate in immune response by recruiting leukocytes to sites of inflammation. VCAM1 helps guide immune cells to directed locations where they perform defense activities. Although VCAM1 is not part of any known large protein complex its interaction with integrins facilitates the migration of immune cells across the endothelium and into tissue.
- Pathways
VCAM1 contributes significantly to the leukocyte extravasation process in inflammatory pathways. It plays a role in both the cytokine-cytokine receptor interaction pathway and the NF-kB signaling pathway which are essential for immune response and cell signaling. Through these pathways it interacts with proteins like ICAM1 and various chemokines helping to coordinate the immune cell movement through vascular tissue barriers.
- Associated diseases and disorders
Abnormal regulation of VCAM1 links to various cardiovascular diseases such as atherosclerosis and inflammatory diseases like rheumatoid arthritis. The protein levels may increase in these conditions promoting excessive immune cell recruitment and tissue damage. VCAM1 also interfaces with other proteins like ICAM1 in these pathological states which together contribute to the progression and severity of these diseases.
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4 product images
Flow Cytometry - Human VCAM1 knockout A549 cell line (ab273758)
Flow cytometry overlay histogram showing wild-type A549 (green line) and VCAM1 knockout A549 cells (red line, ab273758), treated with 10 ng/ml TNF-alpha for 16 h (left) and untreated (right), stained with APC Anti-VCAM1 antibody [STA] ab103173. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (APC Anti-VCAM1 antibody [STA] ab103173) (1x106 in 100μl at 0.2μg/ml) for 30 min at 4°C.
Isotype control antibody mouse IgG1κ Allophycocyanin was used at the same concentration and conditions as the primary antibody (wild-type A549 - black line VCAM knockout A549 - 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 40 mW Red laser (638nm) and 660/10 bandpass filter.
Western blot - Human VCAM1 knockout A549 cell line (ab273758)
Lanes 1 - 6: Merged signal (red and green). Green - Anti-VCAM1 antibody [EPR5047] ab134047 observed at 105 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.Anti-VCAM1 antibody [EPR5047] ab134047 was shown to react with VCAM1 in treated wild-type A549 cells in Western blot with loss of signal observed in treated VCAM1 knockout cell line ab273758 (knockout cell lysate Human VCAM1 knockout A549 cell lysate ab275504). Wild-type A549 and VCAM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-VCAM1 antibody [EPR5047] ab134047 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 ° at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5047] (Anti-VCAM1 antibody [EPR5047] ab134047) at 1/2000 dilution
Lane 1: Wild-type A549 cell lysate at 30 µg
Lane 2: Wild-type A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg
Lane 2: Western blot - Human VCAM1 knockout A549 cell line (ab273758)
Lane 3: VCAM1 knockout A549 cell lysate at 30 µg
Lane 4: VCAM1 knockout A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg
Lane 5: HUVEC cell lysate at 30 µg
Lane 6: HUVEC TNF-a treated (16 ng/mL, 16h) cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 105 kDa
Western blot - Human VCAM1 knockout A549 cell line (ab273758)
Anti-VCAM1 antibody [EPR5038(2)] ab174279 was shown to react with VCAM1 in treated wild-type A549 cells in western blot. Loss of signal was observed when treated VCAM1 knockout cell line ab273758 (knockout cell lysate Human VCAM1 knockout A549 cell lysate ab275504) was used. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-VCAM1 antibody [EPR5038(2)] ab174279 overnight at 4 ° at a 1 in 1000 dilution. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) secondary antibody at 1 in 5000 for 1 hour at room temperature before development with Optiblot ECL reagent (Anti-PKC delta (phospho S299) antibody [EPNCI119] ab133456) and imaging.
All lanes: Western blot - Anti-VCAM1 antibody [EPR5038(2)] (Anti-VCAM1 antibody [EPR5038(2)] ab174279) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 30 µg
Lane 2: Wild-type A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg
Lane 3: VCAM1 knockout A549 cell lysate at 30 µg
Lanes 3 - 4: Western blot - Human VCAM1 knockout A549 cell line (ab273758)
Lane 4: VCAM1 knockout A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 105 kDa
Exposure time: 20s
Sanger Sequencing - Human VCAM1 knockout A549 cell line (ab273758)
Allele-1: 4 bp deletion in exon 2
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