Summary of Contents for PromoKine PK-CA724-488HTS
- Page 1 Cell Proliferation Kit III (EdU; HTS) Instruction Manual Cat.No. PK-CA724-488HTS PK-CA724-555HTS PK-CA724-594HTS* PK-CA724-647HTS*...
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Page 2: Table Of Contents
Instruction Manual Contents Introduction and product description: Materials provided with the Kit and Storage Conditions Required Material and Equipment not included in this Kit Workflow Preparation of the Stock Solutions Labeling of Cells with EdU Staining Cell-surface Antigens with Antibodies (optional) Cell Fixation and Permeabilization EdU Detection Staining Intracellular or Surface Antigens (optional) -
Page 3: Introduction And Product Description
EdU-Click technology protected patents WO2006/117161 WO 03/101972. Any commercial use will require a licence. Please contact us for details. Also available: PromoKine EdU-based Cell Proliferation Assays available for Flow Cytometry (PK-CA724-488FC, PK-CA724-555FC, PK-CA724-594FC & PK-CA724- 647FC) Fluorescence Microscopy (PK-CA724-488FM, PK-CA724-555FM, PK-CA724-594FM &... -
Page 4: Materials Provided With The Kit And Storage Conditions
Table 2: Contents of the kit and storage conditions. Cell Proliferation Kit III (EdU; HTS) ready for 2 x 96 well plate assays: Product number 20X EdU in PBS Used fluorescent dye PK-CA724-488HTS 2 mL 6-FAM Azide (λ =496/λ =516) -
Page 5: Required Material And Equipment Not Included In This Kit
Instruction Manual 2. Required Material and Equipment not included in this Kit Adherent cells Reaction tubes (size depends on the volume of reaction cocktail needed) Buffered saline solution, such as PBS, D-PBS or TBS Fixative solution (4% paraformaldehyde in PBS) ... -
Page 6: Preparation Of The Stock Solutions
Instruction Manual 4. Preparation of the Stock Solutions Allow all vials to warm to room temperature before opening. 4.1.1 For the preparation of a 20 µM stock solution of EdU (2X EdU), add the appropriate amount of aqueous solution (1X PBS) to EdU (component A) according to table 3 and mix until the compound is completely dissolved. -
Page 7: Staining Cell-Surface Antigens With Antibodies (Optional)
Instruction Manual For the desired final concentration, add the appropriate amount of EdU to the culture medium and mix well. We recommend using a concentration of 10 µM for 1-4 hours as a starting point. Use higher EdU concentrations for a shorter incubation time. -
Page 8: Edu Detection
Instruction Manual Remove the wash solution and add to each well 100 µL permeabilization solution. Mix well but gently, incubate for 10-20 minutes at room temperature and proceed to step 8. for the click reaction. 8. EdU Detection Prepare the click assay cocktail in the same order as described in table 5. If the ingredients are not added in the order listed, the reaction will not proceed optimally or might even fail. -
Page 9: Staining Intracellular Or Surface Antigens (Optional)
9, otherwise continue to step 10. 9. Staining Intracellular or Surface Antigens (optional) The PromoKine EdU-based Cell Proliferation Assay (HTS) can be used with antibodies against surface and intracellular markers. To ensure the compatibility of your reagent or antibody, please refer to Table 7. -
Page 10: Example Data
Instruction Manual Excitation Emission Product number Filter (nm) (nm) PK-CA724-488HTS 6-FAM Azide Green PK-CA724-555HTS 5-TAMRA-PEG3-Azide Violet PK-CA724-594HTS* 5/6-Sulforhodamine 101-PEG3-Azide Orange PK-CA724-647HTS* Eterneon Red 645-Azide Table 8: Emission and excitation maxima of the available dyes. * Currently not available. 11. Example of the data derived from an experiment using the EdU-based HTS Kit: Figure 1. - Page 11 Instruction Manual Figure 2: Detection of EdU incorporation via fluorescence microscopy. A fluorescence photograph (40x) of the center of each 96 well of the, with rinse buffer washed assay plate was captured and presented using the Nikon NIS-elements software. Figure 3: Zoom on the samples after Click reaction and washing (in Figure 5) cells which do EdU proliferation in well C9 and cells, which haven’t received EdU, in well G9.
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Page 12: Ordering Information
Instruction Manual Ordering Information Product Name Size Catalog Number Cell Proliferation Kit III (EdU-488; HTS) 200 assays PK-CA724-488HTS Cell Proliferation Kit III (EdU-555; HTS) 200 assays PK-CA724-555HTS Cell Proliferation Kit III (EdU-594; HTS) 200 assays PK-CA724-594HTS* Cell Proliferation Kit III (EdU-647; HTS)
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