StemXVivo EMT Inducing Media Supplement (100X)
R&D Systems, part of Bio-Techne | Catalog # CCM017
Key Product Details
|
Induction of Epithelial to Mesenchymal Transition(EMT) in the A549 human lung carcinoma cell line. The A549 human lung carcinoma cell line was cultured without(A, B); or with(C, D)the StemXVivo®EMT Inducing Media Supplement (Catalog #CCM017) for five days.(A)Control cells exhibit typical epithelial morphology compared to the spindle-shaped, mesenchymal morphology observed in cells following EMT induction(C). EMT status was also assessed by dual immunofluorescence.(B)Untreated cells express the epithelial marker E-Cadherin (red) and exhibit little labeling of the mesenchymal marker Fibronectin (green).D)In contrast, EMT induction results in decreased E-Cadherin expression (red) and an increase in Fibronectin labeling (green). E-Cadherin was detected in cells using a NorthernLights™ (NL) 577-conjugated Goat Anti-Human E-Cadherin Antigen Affinity-purified Polyclonal Antibody (Catalog # NL648R; red). Fibronectin was detected using a Mouse Anti-Human Fibronectin Monoclonal Antibody (Catalog # MAB1918), followed by a NL493-conjugated Donkey Anti-Mouse IgG Secondary Antibody (Catalog # NL009; green). The nuclei were counterstained with DAPI (blue). |
|
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, an epithelial to mesenchymal transition (EMT) is induced using the following in vitro differentiation procedure:
- Add EMT Inducing Media Supplement directly to the standard culture media of your cells of interest
- Culture cells for only 5 days to induce EMT
- Evaluate EMT status using characteristic morphological changes and marker antibodies
Reagents supplied in the StemXVivo® EMT Inducing Media Supplement (Catalog # CCM017):
- 1 mL of StemXVivo® EMT Inducing Media Supplement
Note: The quantity of media supplement is sufficient to drive EMT in the equivalent of fifteen 10 cm diameter tissue culture plates.
Reagents
- Cell culture medium
- Dissociation solution (e.g., TrypLE™ Express, Invitrogen® or equivalent)
- 0.4% Trypan blue solution
Materials
- Epithelial cells of interest
- Tissue culture plates/flasks
- 15 mL centrifuge tubes
- Pipettes and pipette tips
- Serological pipettes
Equipment
- 37 °C and 5% CO2 incubator
- Centrifuge
- Hemocytometer
- Inverted microscope
- 37 °C water bath
- 2 °C to 8 °C refrigerator
Harvest epithelial cells of interest using a dissociation solution.
Transfer the cell suspension to a 15 mL conical tube containing pre-warmed media.
Centrifuge at 400 x g for 5 minutes.
Perform a cell count.
Plate cells at 0.9 - 1.0 x 104 cells/cm2 in media containing 1X EMT Inducing Media Supplement.
Culture cells and monitor cell morphology daily.
Replace media after 3 days.
After 5 days, EMT induction is complete.
Evaluate EMT status using established markers and immunocytochemistry
