Human EMT 3-Color Immunocytochemistry Kit
Human EMT 3-Color Immunocytochemistry Kit Summary
To assess epithelial to mesenchymal transition (EMT) status in human cells using single-step ICC.
Key Benefits
- Provides a straightforward readout of EMT status
- Utilizes single-step, fluorescent ICC staining of EMT markers
- Simultaneous analysis of 3 EMT markers increases confidence in EMT status
Why Assess EMT Status in Human Cells Using Established Markers?
Epithelial to mesenchymal transition (EMT) is centrally important during embryonic development and is implicated in tissue fibrosis and cancer cell metastasis. The ability to quickly and reliably assess EMT status is critical for developing a greater understanding of the mechanisms that regulate EMT.
The downregulation of epithelial markers, including E-Cadherin, and upregulation of mesenchymal markers, including Snail and Vimentin, can be used to assess EMT status.
Assessing EMT status using 3-color ICC:
- Increases confidence in EMT status by combining 3 different fluorochrome-conjugated antibodies for simultaneous staining.
- Allows researchers to evaluate changes in relative protein levels and subcellular localization of EMT markers.
- Ensures efficient use of time and reagents via single-step staining.
This kit contains the following fluorochrome-conjugated antibodies to analyze EMT status in human cells:
- NL557-conjugated Goat Anti-Human Snail
- NL637-conjugated Goat Anti-Human E-Cadherin
- NL493-conjugated Goat Anti-Human Vimentin
Each antibody is supplied as a 10X stock; enough for 25 assays when used in a 50 µL staining volume per assay.
Stability and Storage
Reagents are stable for 12 months from date of receipt when stored in the dark at 2° C to 8°.
Specifications
Product Datasheets
Scientific Data
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Upregulation of the Mesenchymal Markers, Snail and Vimentin, in EMT-induced Cells A549 human lung carcinoma and MCF 10A human breast epithelial cells were either untreated or cultured with the StemXVivo®EMT Inducing Media Supplement (Catalog # CCM017) for five days. The cells were analyzed for an epithelial to mesenchymal transition (EMT) by simultaneously staining with the antibodies contained in the Human EMT 3-Color Immunocytochemistry Kit (Catalog # SC026): NorthernLights™ (NL)637-conjugated Goat Anti-Human E-Cadherin (pseudocolored white), NL557-conjugated Goat Anti-Human Snail (red), and NL493-conjugated Rat Anti-Human Vimentin (green). Cells cultured with the EMT Inducing Media Supplement showed downregulation of the epithelial marker, E-Cadherin, and concurrent upregulation of the mesenchymal markers, Snail and Vimentin, compared to control cells.
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, epithelial to mesenchymal transition (EMT) status in human cells can be analyzed via 3-Color ICC using the following protocol:
- EMT is induced in cells of interest
- Fluorochrome-conjugated primary antibodies are added to fixed cells
- EMT markers are visualized using a fluorescence microscope
Reagents supplied in the Human EMT 3-Color Immunocytochemistry Kit (Catalog # SC026):
- NL557-conjugated Goat Anti-Human Snail
- NL637-conjugated Goat Anti-Human E-Cadherin
- NL493-conjugated Goat Anti-Human Vimentin
This kit contains sufficient reagents to process up to 1 x 109 total cells.
Reagents
- Cells
- Coverslips (sterilized)
- Cell culture plate (24-well)
Materials
- Human pluripotent stem cells
- Pipettes and pipette tips
- Serological pipettes
Equipment
- 37 °C, 5% CO2 incubator
- Centrifuge
- Hemocytometer
- Inverted microscope
- Fluorescence microscope
Plate human epithelial cells.
Culture under desired EMT-inducing conditions.
Fix cells with 4% paraformaldehyde.
Block in blocking solution.
Incubate with fluorochrome-conjugated primary antibodies.
Wash with wash buffer.
Incubate with nuclear counterstain.
Visualize using a fluorescence microscope and appropriate filter sets
Citations for Human EMT 3-Color Immunocytochemistry Kit
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Assessing metastatic potential of breast cancer cells based on EGFR dynamics
Authors: YL Liu, CK Chou, M Kim, R Vasisht, YA Kuo, P Ang, C Liu, EP Perillo, YA Chen, K Blocher, H Horng, YI Chen, DT Nguyen, TE Yankeelov, MC Hung, AK Dunn, HC Yeh
Sci Rep, 2019-03-04;9(1):3395. 2019-03-04
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Oncostatin M induces tumorigenic properties in non-transformed human prostate epithelial cells, in part through activation of signal transducer and activator of transcription 3 (STAT3)
Authors: S Sterbova, T Karlsson, E Persson
Biochem. Biophys. Res. Commun., 2018-03-12;0(0):. 2018-03-12
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