Vala Sciences

PKC-Alpha Screen-Certified Cell-Imaging Kit   (Z’ > 0.5)

Overview

Protein kinase Cα(PKCα), a key regulatory protein in signal transduction pathways modulating cancer and inflammation, is activated via G-protein coupled receptors, tumor promoting chemicals, and other stimuli. Activation of PKCa depends upon migration of PKCa from the cytoplasm to the cell membrane. The expression and activation of this important signal transduction molecule can be quantified in cultured cells (validated for HeLa, UMR106, or IEC18 cells), with Vala Sciences Inc's PKCa kit.

Item number: 4801

Kit Applications

• Monitoring Activation of Protein Kinase C-alpha via HT Imaging (Z’ > 0.5)

Kit Usage & Contents

Thank you for purchasing the Protein kinase Cα activation assay kit from Vala Scences Inc. Our product provides an exceptional combination of reagents and software tools to enable our customers to screen for potential activators or inhibitors of Protein kinase Cα, a protein that plays a key role in many forms of cancer.

• Store kit components at 4°CFor storage >30 days, freeze Reagent A in aliquots.
• Components B - F contain 0.02% sodium azide (NaN3). Dispose of properly! 
• Flush plumbing with excess water to avoid gas build-up. 

Kit Components:

 

• Reagent A (K001/A): Fixative (60 ml). (CAUTION: CONTAINS 4% PARAFORMALDEHYDE - OPEN AND USE IN A WELL VELTILATED SPACE. AVOID CONTACT AND DISPOSE OF PROPERLY)
• Reagent B (K001/B): (60 ml)
• Reagent C: (K001/C) (60 ml)
• Reagent D(K001/D): (18 ml base solution; 180 μl of 100X stock) - Only prepare enough reagent D for the experiment, as it is best to store it in it's undiluted form at 4 C. Prepare dilution of Reagent D by adding 10 μl of 100X 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 E (K001/E): (18 ml base solution; 72 μl of 250X stock) - Only prepare enough reagent E for the experiment, as it is best to store it in it's undiluted form at 4 C. Prepare dilution of Reagent E by adding 4 μl of 250X 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 F (K001/F): Nuclear stain (60 ml) (CAUTION: CONTAINS DAPI - AVOID CONTACT AND DISPOSE OF PROPERLY)
• 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:

 

• Cells (e.g., HeLa, UMR106, or IEC18 cells)
• Phosphate buffered saline (PBS): (e.g., MediaTech Cat. No. 21-040-CM, PBS, 1X).

 

Protocol: The following protocol has been optimized to visualize PKCα in HeLa cells (ATTC CCL-2, human cervical cancer). Application of this protocol to other cell types may require slight modifications. Reagents in this kit can detect and quantify PKCα from human, rat, or mouse. The key steps are preparing the cells for the screen, performing the screen, processing the samples, imaging the cells, analyzing the data.

1. Preparing the cells for the screen: 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., Matrigel, BD Biosciences, diliuted 1/50 using "thin-coat method" as specified by manufacturer). Plate 1 to 1.5 x 104 cells per well in growth media (DMEM+10% FBS), allow the cells to proliferate to confluence (24 to 48 hr), then switch the cells to DMEM+2% FBS 48 hr prior to the experiment. Refeed cells with DMEM+2% FBS 2 to 4 hr prior to performing the experiment.

2. Prepare the reagents: Cool PBS (at least 21 ml per plate) to 4 C. Prepare test compounds (these can be prepared as either concentrated stock solutions, e.g., 10X, or at the desired final concentration (1X, which will require a full replacement of the well contents with the solution containing the test compound). Prepare a station for fixing the cells in a well ventilated place (e.g., take the bottle of Reagent A to the fume hood, setup reagent reservoirs for PBS and for Reagent A).

3. Perform the experiment: Tests for PKC activation are typically carried out with drug incubations from 5 to 30 mins.
A) Add test compounds: If drug solutions are at 1X, dump or aspirate each plate and replace well contents with test conditions. If concentrated drug stock solutions are being used, add these to the cells at this time.
B) Incubate cells for the desired period of time (e.g., 5 to 30 mins): Final preparations for "stopping" the cells can be made at this time (e.g., pipetting PBS and Reagent A into separate reagent reservoirs).
C) "Stop" the 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 (100 μl/well) for 15 minutes at room temperature to "fix" the cells. Rinse wells 2X with 200 μl of PBS to remove fixative.

4. Stain the cells: 
- Add 100 μl/well Reagent B. Incubate at 37 C for 15 mins with rotation. Remove Reagent B.
- Add 100 μl/well Reagent C. Incubate at 37 C for 60 mins with rotation. Remove Reagent C (remove as much as possible from each well, using a multi-channel pipet or aspirator, being careful not to disturb the cell monolayer).
- Add 30 μl/well Reagent D. Incubate at 37 C for 60 mins with rotation.
- Incubate wells with 200 μl/well PBS at 37 C for 5 mins with rotation. Repeat for a total of 3 rinses. Remove PBS.
- Add 30 μl/well of Reagent E. Incubate at 37 C for 60 mins with rotation.
- Incubate wells with 300 μl/well PBS at 37 C for 5 mins with rotation. Repeat for a total of 3 rinses. Remove PBS.
- Add 100 μl/well of Reagent F. Incubate 20 minutes at room temperature. The cells are now ready for imaging.

5. Imaging the cells: Refer to the manual of your microscopy work station for image acquisition. Avoid overexposing the images. We typically image HeLa cells using either a 20X or 40X objective. Sharp focus is critical! PKCα will be visualized on the "green" channel (excitation at 490 nm, emitting at 520 nm). Nuclei will be visualized on the "blue" channel (excitation filter centered at 360 nm, emitting at 460 nm).

Technical tips:

The Vala Sciences PKCα activation software performs best on cells that are in a confluent monolayer. There should be no significant gaps between cells or areas where cells have "piled up".

If desired, cells can be stored at 4 C in Reagent C for up to 3 days prior to further processing.

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.

« Reagents