Cell Proliferation Kit III (EdU; HTS)
Instruction Manual
Cat.No. PK-CA724-488HTS
PK-CA724-555HTS
PK-CA724-594HTS*
PK-CA724-647HTS*
Страница 1: ...Cell Proliferation Kit III EdU HTS Instruction Manual Cat No PK CA724 488HTS PK CA724 555HTS PK CA724 594HTS PK CA724 647HTS ...
Страница 2: ...and Equipment not included in this Kit 5 Workflow 5 Preparation of the Stock Solutions 6 Labeling of Cells with EdU 6 Staining Cell surface Antigens with Antibodies optional 7 Cell Fixation and Permeabilization 7 EdU Detection 8 Staining Intracellular or Surface Antigens optional 9 Imaging and analysis 9 Example Data 10 Ordering Information 12 ...
Страница 3: ...rated nucleoside Instead the EdU based Cell Proliferation Assays utilize click chemistry for detection in a variety of dye fluorescent readouts Furthermore the streamlined detection protocol reduces both the total number of steps and significantly decreases the total amount of time The simple click chemistry detection procedure is complete within 30 minutes and is compatible with multiplexing for ...
Страница 4: ...or at least one year When dissolved the component E has to be kept at 20 C for long term storage Cautions The rinse buffer Component F contains hazardous components Use with appropriate precautions Keep away from acids to avoid dangerous gases Handle reagents containing the rinse buffer using equipment and practices appropriate for the hazards posed by such materials Use gloves Dispose of the reag...
Страница 5: ...re many factors which can influence the labeling such as the growth medium the density and the type of cells To determine the optimal concentration for your experiment a range of EdU concentrations should be tested for your cell type and experimental conditions Principally a similar concentration to BrdU can be used for EdU as a starting point Heparin can be used as anticoagulant for collection if...
Страница 6: ...ore any remaining solution at 20 C When stored as directed this stock solution is stable for up to 6 months We recommend preparing aliquots to avoid repeated thaw and freeze cycles EdU based HTS Kit Buffer additive solide Dilution volume of deionized water 1 well plate 100 mg 1 mL 2 x 96 well plates 200 mg 2 5 mL Table 4 Amounts of aqueous solution needed to dissolve the buffer additive to the fin...
Страница 7: ...ix well but gently Note PE PE tandem or Quantum Dot antibody conjugates should not be used before performing the click reaction step 8 6 5 Incubate the cells for the recommended length of time and temperature Protect from light 6 6 Proceed to step 7 7 Cell Fixation and Permeabilization This protocol was developed with a fixation step using 4 Paraformaldehyde in PBS followed by permeabilization ste...
Страница 8: ...ail to each well and mix well but gently to distribute the assay solution evenly 8 3 Incubate the click assay mixture for 30 minutes at room temperature Protect from light 8 4 From the 10x rinse solution prepare a 1x rinse solution by applying following table table 6 Add the appropriate amount of PBS 1X see table 6 to the component F and mix well To prevent crystallization keep component F at room...
Страница 9: ...anin APC and APC based tandems Compatible R phycoerythrin R PE and R PE based tandems Use R PE and R PE based tandems after the EdU detection reaction Quantum Dots Use Quantum Dots after the EdU detection reaction Fluorescent proteins e g GFP Use anti GFP antibodies before the EdU detection reaction or use organic dye based reagents for protein expression detection Table 1 EdU detection dye compat...
Страница 10: ...cated cell numbers per well After 42 hours cells were incubated with or without 10 μM EdU for 2 4 or 6 h and subsequently EdU incorporation was detected using the EdU based Cell Proliferation Assays III EdU HTS and a fluorescence plate reader Mean and SD values from quadruplicates are shown Product number Dye Excitation nm Emission nm Filter PK CA724 488HTS 6 FAM Azide 496 516 Green PK CA724 555HT...
Страница 11: ...raph 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 ...
Страница 12: ...00 assays PK CA724 647HTS PromoCell GmbH Sickingenstr 63 65 69126 Heidelberg Germany USA Canada Phone 1 866 251 2860 toll free Fax 1 866 827 9219 toll free Deutschland Telefon 0800 776 66 23 gebührenfrei Fax 0800 100 83 06 gebührenfrei France Téléphone 0800 90 93 32 ligne verte Téléfax 0800 90 27 36 ligne verte United Kingdom Phone 0800 96 03 33 toll free Fax 0800 169 85 54 toll free Other Countri...
