Human CCL2 (MCP1) knockout HeLa cell line
- Advanced Validation
- What is this?
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(0 Publication)
- ICC
Lab
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).
- WB
Lab
Western blot - Human CCL2 (MCP1) knockout HeLa cell line (AB255372)
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.
All lanes:
Western blot - Anti-MCP1 antibody [EPR21025] (<a href='/en-us/products/primary-antibodies/mcp1-antibody-epr21025-ab214819'>ab214819</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa Vehicle Control TNF alpha (0ng/mL, 6h) + Brefeldin A (1μg/ml,3h) cell lysate at 20 µg
Lane 2:
Wild-type HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1μg/ml,3h) cell lysate at 20 µg
Lane 3:
CCL2 knockout HeLa Vehicle Control TNF alpha (0ng/mL, 6h) + Brefeldin A (1μg/ml,3h) cell lysate at 20 µg
Lane 4:
CCL2 knockout HeLa Treated TNF alpha (20ng/mL, 6h) + Brefeldin A (1μg/ml,3h) cell lysate at 20 µg
Predicted band size: 11 kDa
Observed band size: 11 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human CCL2 (MCP1) knockout HeLa cell line (AB255372)
Allele-1 : 5 bp deletion in exon 2.
- Sanger seq
Unknown
Sanger Sequencing - Human CCL2 (MCP1) knockout HeLa cell line (AB255372)
Allele-2 : 1 bp insertion in exon 2.
Complementary Products
Primary Antibodies
AB214819
BOND RX™ Validated|RabMAb|Recombinant|KO Validated
![Immunocytochemistry - Anti-MCP1 antibody [EPR21025] (AB214819)](https://content.abcam.com/products/images/mcp1-antibody-epr21025-ab214819--immunocytochemistry-img172752.jpg)
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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
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.
Culture medium
DMEM (High Glucose) + 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
The role of MCP-1 is significant in guiding immune cells to areas requiring immune response. It does not form part of a complex but operates as a singular entity to affect immune signaling. MCP-1 influences the behavior of monocytes and macrophages by inducing chemotaxis and activating their effector functions. Through this mechanism it aids in the inflammatory response and tissue repair.
Pathways
MCP-1 participates in the inflammatory pathways by cooperating with key proteins like CCR2. It facilitates the JAK-STAT signaling pathway which is vital for transmitting extracellular information into cellular responses. The interaction of MCP-1 with CCR2 influences the activation of downstream proteins like STAT3 enabling various immune responses. MCP-1 thereby plays a central role in modulating inflammation-related pathways.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com