E-Cadherin Screen-Certified Cell-Imaging Kit (Z’ > 0.5)
$700.00
Availability: In Stock • Product size: 500 tests using 96 well plates
Overview
E-cadherin, a cell-cell adhesion molecule, is frequently downregulated in a wide variety of tumors (breast, skin, prostate, gastric, and colon cancers), suggesting that it is an important tumor suppressor. E-cadherin is the major binding partner for -catenin, and E-cadherin downregulation is often accompanied by beta-catenin translocation to the nucleus, and the stimulation of cell division. E-cadherin expression and localization can be quantified in cultured cells with Vala Sciences Inc's E-cadherin kit.
Item number: 4803
Kit Applications
Kit Usage & Contents
- Store kit components at 4°C
- For storage >30 days, freeze Reagent A in aliquots appropriate for your experiments.
- Components B - D contain 0.02% sodium azide (NaN3). Dispose of properly! Flush plumbing with excess water to avoid gas build-up.
Technical tip! Adipocytes are sometimes poorly adherent to culture dishes. Rinse gently to avoid losing cells.
Kit Components:
- Reagent A: Fixative (60 ml). (CAUTION: CONTAINS 4% PARAFORMALDEHYDE - OPEN AND USE IN A WELL VELTILATED SPACE. AVOID CONTACT AND DISPOSE OF PROPERLY)
- Reagent B: Permeabilizer (60 ml)
- Reagent C: Lipid stain (18 ml base solution; 60 μl of 333X stock) - Only prepare enough reagent D for the experiment, as it is best to store it in it's undiluted form at -4 C. Vortex Reagent C 333X stock solution thoroughly. Prepare dilution of Reagent C by adding 3 μl of 333X stock solution per ml of base solution, just prior to use. Enough reagent is supplied to use 30 μl per well for five 96-well dishes.
- Reagent D: Nuclear stain (60 ml) (CAUTION: CONTAINS DAPI - AVOID CONTACT AND DISPOSE OF PROPERLY)
- These reagents have been validated by Vala Sciences Inc on human adipocytes, adipocytes derived from murine 3T3-L1 cells, and HeLa cells cultured in media containing fatty acids.
- Software: The latest version of CyteSeer, Vala Sciences' Automated Cell Analysis Software, can be downloaded from http://www.valasciences.com. A time limited license, unlocking CyteSeer's full capabilities, is automatically generated for any new workstation. CyteSeer is free for ongoing use as a viewer of both microscope images and CyteSeer generated cell-by-cell image mask & numeric data. Contact Vala directly for licensing details on continued automated cell analysis capabilities.
- Algorithm: Membrane Segmentation
Reagents that are required but are not supplied with the kit include:
- Phosphate buffered saline (PBS): (e.g., MediaTech Cat. No. 21-040-CM, PBS, 1X).
Protocol: The key steps are preparing the cells for the screen, performing the screen, staining the cells, imaging the cells, analyzing the data.
1. Preparing the cells: Use 96-well dishes featuring optical quality coverslip glass (e.g., Nunc #164588) for best results. Prior to plating, pre-coat dishes with a protein substrate (e.g., 1% gelatin that has been crosslinked with glutaraldehyde and extensively rinsed - protocol available upon request). Plate cells and allow to grow to near confluency. If appropriate, add media supplements to induce lipid droplet formation (for 3T3-L1 cells, culture cells several days post-confluency in DMEM +10% FBS, then supplement with 0.5 mM IBMX, 0.25 μM dexamethasone, and 1 μg/ml insulin for 3 to 4 days, then switch back to the base medium; lipid droplets will form over the next 5 days).
2. Performing the screen: Remove base culture medium and replace with culture medium containing test compounds. Altering the number and size of the lipid droplets is likely to require exposure times of 1 to 7 days. Positive controls that induce extensive lipid droplet formation in adipocytes include thiazolidinediones (such as rosiglitazone).
3. Stain the cells. For automated analysis of lipid droplet size and number it is necessary to stain both the lipid droplets and the nuclei.
- Fix cells: Remove test solutions, rinse wells with 200 μl/well of cold PBS, add 100 μl of Reagent A per well. Incubate the cells with Reagent A for 15 minutes at room temperature. Rinse wells 1X with 200 μl of PBS to remove fixative.
- Permeabilize cells: Add 100 μl/well Reagent B. Incubate at 37 C for 15 mins with rotation. Remove Reagent B.
- Stain the lipid droplets: Add 100 μl/well Reagent C. Incubate at 37 C for 60 mins with rotation. Remove Reagent C. Rinse 3X with PBS.
- Stain nuclei: Add 100 μl/well of Reagent D. Incubate 20 minutes at room temperature. The cells are now ready for imaging.
4. Imaging the cells: Refer to the manual of your microscopy work station for image acquisition. Avoid overexposing the images. We typically image adipocytes using a 40X objective. Sharp focus is critical! For each field of view, make sure to image the cells in two fluorescence channels (blue and green). Visualize nuclei on the "blue" channel (excitation filter centered at 360 nm, emitting at 460 nm). Visualize lipid droplets on the "green" channel (excitation at 490 nm, emitting at 520 nm).
5. Quantifying lipid droplet number and size: See instruction manual and tutorials in Vala Sciences CyteSeer software manual.
Ordering
In addition to ordering online you can also call us on 1-888-742-8252 (VALA) or fax an order form to 1-888-742-1230. If you have any questions about this item you can also contact us through the website. Items that are in stock will be delivered in 1-2 days.
CyteSeer
Process your cell images with CyteSeer, our automated high-content analysis software.
