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PromoKine PK-CA724-488HTS, Инструкция по эксплуатации

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Instruction Manual 

Introduction and product description: 

The detection of cell proliferation is of utmost importance for assessing cell health, 
determining genotoxicity or evaluating anticancer drugs. This is normally performed by 
adding nucleoside analogs like [

3

H]thymidine or 5-bromo-2’-deoxyuridine (BrdU) to 

cells during replication, and their incorporation into DNA is detected or visualized by 
autoradiography or with an anti-BrdU-antibody respectively. Both methods exhibit 

several limitations. Working with [

3

H]thymidine is troublesome because of its 

radioactivity. Autoradiography is slow and thus not suitable for rapid high-throughput 
studies. The major disadvantage of BrdU staining is that the double-stranded DNA 
blocks the access of the anti-BrdU antibody to BrdU units. Therefore, samples have to 
be subjected to harsh denaturing conditions resulting in degradation of the structure of 
the specimen. 
 
The 

PromoKine EdU-based Cell Proliferation Assays 

overcome these limitations, 

providing a superior alternative to BrdU and [

3

H]thymidine assays for directly measuring 

DNA synthesis of adherent cells in 96 well plates. EdU (5-ethynyl-2’-deoxyuridine) is a 

nucleoside analog to thymidine and is incorporated into DNA during active DNA 
synthesis. In contrast to BrdU assays, the 

EdU-based Cell Proliferation Assays 

are not 

antibody based and therefore do not require DNA denaturation for detection of the 
incorporated 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 content and 
context-rich results. 

 
For research use only.  
 

Information in this document is subject to change without notice. PromoCell GmbH 

assumes no responsibility for any errors that may appear in this document. 
PromoCell GmbH disclaims all warranties with respect to this document, expressed or 
implied, including but not limited to those of merchantability or fitness for a particular 
purpose. In no event shall PromoCell  GmbH be liable, whether in contract, tort, 
warranty, or under any statute or on any other basis for special, incidental, indirect, 
punitive, multiple or consequential damages in connection with or arising from this 
document, including but not limited to the use thereof. 
 
Please read the material safety data sheets (MSDS) provided for each 
product/component. 

 
The EdU-Click technology is protected by  patents  WO2006/117161 and 

 

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)  and 

Fluorescence Microscopy

  (PK-CA724-488FM, PK-CA724-555FM,  

PK-CA724-594FM & PK-CA724-647FM) are also available. 
See our website for more information. 

Содержание PK-CA724-488HTS

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...

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