Human VCAM1 knockout A549 cell line (ab273758)
Overview
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Product name
Human VCAM1 knockout A549 cell line -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 4 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WB, ICCmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type A549 cell line (ab275463). 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 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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Lung -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
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
Important in cell-cell recognition. Appears to function in leukocyte-endothelial cell adhesion. Interacts with the beta-1 integrin VLA4 on leukocytes, and mediates both adhesion and signal transduction. The VCAM1/VLA4 interaction may play a pathophysiologic role both in immune responses and in leukocyte emigration to sites of inflammation. -
Tissue specificity
Expressed on inflammed vascular endothelium, as well as on macrophage-like and dendritic cell types in both normal and inflammed tissue. -
Sequence similarities
Contains 7 Ig-like C2-type (immunoglobulin-like) domains. -
Domain
Either the first or the fourth Ig-like C2-type domain is required for VLA4-dependent cell adhesion. -
Post-translational
modificationsSialoglycoprotein. -
Cellular localization
Membrane. - Information by UniProt
Associated products
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Recombinant Protein
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Related Products
- APC Anti-VCAM1 antibody [STA], prediluted (ab103173)
- Anti-VCAM1 antibody [EPR5047] (ab134047)
- Anti-VCAM1 antibody [EPR5038(2)] (ab174279)
- Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (ab215380)
- Anti-VCAM1 antibody [EPR5038(2)] - BSA and Azide free (ab249790)
- Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (ab271899)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab273758 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.
|
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| ICC |
Use at an assay dependent concentration.
|
| Notes |
|---|
|
WB
Use at an assay dependent concentration. |
|
ICC
Use at an assay dependent concentration. |
Images
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Western blot - Human VCAM1 knockout A549 cell line (ab273758)All lanes : Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/2000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : Wild-type A549 TNF-a treated (10 ng/mL, 16h) cell lysate
Lane 3 : VCAM1 knockout A549 cell lysate
Lane 4 : VCAM1 knockout A549 TNF-a treated (10 ng/mL, 16h) cell lysate
Lane 5 : HUVEC cell lysate
Lane 6 : HUVEC TNF-a treated (16 ng/mL, 16h) cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Observed band size: 105 kDa why is the actual band size different from the predicted?Lanes 1 - 6: Merged signal (red and green). Green - ab134047 observed at 105 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
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 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 ab134047 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C 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 (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
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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 ab103173. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (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.
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Western blot - Human VCAM1 knockout A549 cell line (ab273758)All lanes : Anti-VCAM1 antibody [EPR5038(2)] (ab174279) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : Wild-type A549 TNF-a treated (10 ng/mL, 16h) cell lysate
Lane 3 : VCAM1 knockout A549 cell lysate
Lane 4 : VCAM1 knockout A549 TNF-a treated (10 ng/mL, 16h) cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Observed band size: 105 kDa why is the actual band size different from the predicted?
Exposure time: 20 secondsab174279 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 ab275504) was used. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab174279 overnight at 4 °C 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 (ab133456) and imaging.
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Sanger Sequencing - Human VCAM1 knockout A549 cell line (ab273758)Allele-1: 4 bp deletion in exon 2
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab273758 has not yet been referenced specifically in any publications.