Human CCL2 (MCP1) knockout HeLa cell line (ab255372)
Overview
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Product name
Human CCL2 (MCP1) knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and 5 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing -
Tested applications
Suitable for: ICC, WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HeLa cell line (ab255928). 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: DMEM (High Glucose) + 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 2x104 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 2x104 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.
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10 -
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
Chemotactic factor that attracts monocytes and basophils but not neutrophils or eosinophils. Augments monocyte anti-tumor activity. Has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, like psoriasis, rheumatoid arthritis or atherosclerosis. May be involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis. -
Sequence similarities
Belongs to the intercrine beta (chemokine CC) family. -
Post-translational
modificationsProcessing at the N-terminus can regulate receptor and target cell selectivity. Deletion of the N-terminal residue converts it from an activator of basophil to an eosinophil chemoattractant. -
Cellular localization
Secreted. - Information by UniProt
Associated products
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab255372 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 |
|---|---|---|
| ICC |
Use at an assay dependent concentration.
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| WB |
Use at an assay dependent concentration.
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| Notes |
|---|
|
ICC
Use at an assay dependent concentration. |
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WB
Use at an assay dependent concentration. |
Images
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Western blot - Human CCL2 (MCP1) knockout HeLa cell line (ab255372)All lanes : Anti-MCP1 antibody [EPR21025] (ab214819) at 1/1000 dilution
Lane 1 : Wild-type HeLa Vehicle Control TNF alpha (0ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lane 2 : Wild-type HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lane 3 : CCL2 knockout HeLa Vehicle Control TNF alpha (0ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lane 4 : CCL2 knockout HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1µg/ml,3h) cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 11 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-MCP1 antibody [EPR21025] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab214819 was shown to bind specifically to MCP1. A band was observed at 11 kDa in wild-type HeLa cell lysates with no signal observed at this size in ccl2 knockout cell line ab255372 (knockout cell lysate ab263807). To generate this image, wild-type and ccl2 knockout 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-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunocytochemistry - Human CCL2 (MCP1) knockout HeLa cell line (ab255372)ab214819 staining MCP1 in wild-type and MCP1 knockout HeLa cells (ab255372), untreated or treated with TNF-alpha (20ng/ml, 6 hours) and Brefeldin A (1μg/ml, 3 hours). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab214819 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Sanger Sequencing - Human CCL2 knockout HeLa cell line (ab255372)Allele-2: 1 bp insertion in exon 2.
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Sanger Sequencing - Human CCL2 knockout HeLa cell line (ab255372)Allele-1: 5 bp deletion in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab255372 has not yet been referenced specifically in any publications.