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- Additional protein information
- Analytical tools
CellSimple™ Epithelial to Mesenchymal Transition Antibody Assay Kit #15472
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CellSimple™ cell-based analysis of MCF7 (left panel) and MDA-MB-231 (right panel) cells using the CellSimple™ Epithelial to Mesenchymal Transition Antibody Assay Kit. Data was collected in both green (525/45 nm) and red channels (561 nm/LP) and analyzed on the Open Flow Cytometry application. Mean fluorescence intensity (MFI) of E-Cadherin (24E10) Rabbit mAb (Alexa Fluor® 488 Conjugate) #3199 (x-axis) and Vimentin (D21H3) XP® Rabbit mAb (PE Conjugate) #12020 (y-axis) is shown in each dot plot. Cell concentration, MFI, and percentages are displayed in the boxes adjacent to each quadrant. Instrument screen shots are shown.Learn more about how we get our images
Gallery: CellSimple™ Epithelial to Mesenchymal Transition Antibody Assay Kit #15472
- CellSimple™ cell-based analysis of MCF7 (left panel) and MDA-MB-231 (right panel) cells using the CellSimple™ Epithelial to Mesenchymal Transition Antibody Assay Kit. Data was collected in both green (525/45 nm) and red channels (561 nm/LP) and analyzed on the Open Flow Cytometry application. Mean fluorescence intensity (MFI) of E-Cadherin (24E10) Rabbit mAb (Alexa Fluor® 488 Conjugate) #3199 (x-axis) and Vimentin (D21H3) XP® Rabbit mAb (PE Conjugate) #12020 (y-axis) is shown in each dot plot. Cell concentration, MFI, and percentages are displayed in the boxes adjacent to each quadrant. Instrument screen shots are shown.
Immunostaining Protocol for CellSimple™ Antibody-based Assay for Kits
These kits were specially designed for use with the CellSimple™ Cell Analyzer. However, they may also be used with a flow cytometer or plate reader capable of providing excitation between 480 nm and 490 nm and detecting fluorescent emission between 520 nm and 590 nm.
B. Kit components:
- Antibody pair (kit specific)
- 16% Formaldehyde (Methanol-free)
NOTE: The screw cap allows for the entire vial contents to be used at once. To extend the product's shelf life, small volumes should be extracted by piercing the silicone top with a needle and syringe. Store protected from light and use within one month after opening.
- Phosphate Buffered Saline (PBS-20X)
C. Additional reagents needed, but not supplied.
- 90% methanol
- Bovine Serum Albumin #9998 or equivalent
- Reverse osmosis/deionized (RO/DI) water or equivalent
D. Reagent preparation
- 1X PBS: To prepare 500 ml 1X PBS add 25 ml PBS-20X to 475 ml RO/DI water, mix.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin in 100 ml 1X PBS. Store at 4°C.
- Collect cells by centrifugation and aspirate supernatant. For adherent cells, remove cells from the plate or flask by trypsinization followed by trypsin neutralization with medium that contains 10% fetal bovine serum.
- Resuspend cells in 0.5 - 1 ml 1X PBS to 0.5 X 106 - 5 X 106 cells/ml by gently pipetting up and down. Add 16% Formaldehyde to obtain a final concentration of 4% and vortex gently (for example 335 µl of 16% Formaldehyde added to 1 ml of cell suspension).
- Fix for 10 min at 37°C.
- Chill tubes on ice for 1 minute.
- Spin down the cells at 400 x g for 5 minutes in a chilled microcentrifuge.
- Carefully remove the supernatant containing formaldehyde into a hazardous waste receptacle.
- Permeabilize cells by slowly adding 1 - 1.5 ml of ice-cold 90% methanol. Pipette up and down several times to ensure uniform cell suspension.
- Incubate 30 min on ice.
- Proceed with immunostaining or store cells at -20°C in 90% methanol.
- Aliquot 0.5 - 1 x 106 cells into each assay tube.
- Wash the cells twice in 1 ml incubation buffer by centrifugation at 400 x g for 5 minutes.
- Dilute each of the two fluorochrome-conjugated antibodies 1:50 in incubation buffer. Prepare enough diluted antibody mix to resuspend each of your test samples in 25 µl. For example, for 3 test samples add 1.5 µl of antibody mix to 75 µl of incubation buffer. Note: the antibodies are mixed together to allow a simultaneous detection in both the green and the red channels.
- Add 25 µl of the diluted antibody mix to each test sample and gently pipette up and down a few times to ensure even cell suspension.
- Incubate for 1 hour at room temperature with occasional gentle mixing.
- Wash once by centrifugation using incubation buffer.
- Resuspend cells in 0.1 - 0.2 ml PBS and analyze 75 µl on CellSimple™ Cell Analyzer using the Open Flow Cytometry application and using both Green (525/45 nm) and Red (561 nm LP) channels. For more information on how to use the Open Flow Cytometry application and detailed instructions on how to operate the CellSimple™ Cell Analyzer please refer to the CellSimple™ user guide.
posted June 2016
revised November 2016
|Product Includes||Quantity (with Count)|
|Vimentin (D21H3) XP® Rabbit mAb (PE Conjugate) 12020||1 x 25 µl|
|E-Cadherin (24E10) Rabbit mAb (Alexa Fluor® 488 Conjugate) 3199||1 x 25 µl|
|16% Formaldehyde, Methanol-Free 12606||2 x 10 ml|
|Phosphate Buffered Saline (PBS-20X) 9808||1 x 25 ml|
CellSimple™ Epithelial to Mesenchymal Transition Antibody Assay Kit is a fluorescent assay designed for use with the CellSimple™ Cell Analyzer. The kit includes E-Cadherin (24E10) Rabbit mAb (Alexa Fluor® 488) #3199 and Vimentin (D21H3) XP® Rabbit mAb (PE Conjugate) #12020, that can differentiate between epithelial and mesenchymal cell origins, respectively. Thus mean fluorescence intensity (MFI) in the green (525/45 nm) and red (561 nm LP) channels can be used as an indicator for cells undergoing epithelial-mesenchymal transition (EMT).
Antibodies provided in the CellSimple™ Epithelial to Mesenchymal Transition Antibody Assay Kit detect endogenous levels of their respective target.Species Reactivity: Human
The CellSimple™ Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.
Epithelial-mesenchymal transition (EMT) is an essential process during development whereby epithelial cells aquire mesenchymal, fribroblast-like properties and display reduced intracellular adhesion and increased motility. This is a critical feature of normal embryonic development, which is also utilized by malignant epithelial tumors to spread beyond their origin (1-3). This tightly regulated process is associated with a number of cellular and molecular events. EMT depends on a reduction in expression of cell adhesion molecules. Cadherins mediate calcium-dependent cell-cell adhesion and play critical roles in normal tissue development (4). E-cadherin is considered to be an active suppressor of invasion and growth in many epithelial cancers (4-6). Recent studies indicate that cancer cells have upregulated N-cadherin in addition to loss of E-cadherin. This change in cadherin expression is called the "cadherin switch" and downregulation of E-cadherin is one of the hallmarks of EMT (1). Cells undergoing EMT may also continue to express epithelial markers and show epithelial morphology while exhibiting increased expression of mesenchymal markers such as vimentin (7).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. CellSimple is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).