USER MANUAL EdU Click HTS
Contents
1. 12 ROTI kit for High Throughput Screening Ordering information for detailed kit content see Table 2 Carl Roth GmbH Co KG ROTI kits for High Throughput Screening for 2 x 96 well plate assays Product number Product Used fluorescent dye 7786 1 EdU Click HTS 2 488 6 FAM Azide 7789 1 EdU Click HTS 2 555 5 TAMRA PEG3 Azide 7793 1 EdU Click HTS 2 594 5 6 Sulforhodamine 101 PEG3 Azide 7796 1 EdU Click HTS 2 647 Eterneon Red 645 Azide Cyanine 5 Azide analogue ROTI kits for High Throughput Screening for 4 x 96 well plate assays Product number Product Used fluorescent dye 7787 1 EdU Click HTS 4 488 6 FAM Azide 7791 1 EdU Click HTS 4 555 5 TAMRA PEG3 Azide 7794 1 EdU Click HTS 4 594 5 6 Sulforhodamine 101 PEG3 Azide 7797 1 EdU Click HTS 4 647 Eterneon Red 645 Azide Cyanine 5 Azide analogue ROTI kits for High Throughput Screening for 20 x 96 well plate assays Product number Product Used fluorescent dye 7788 1 Pau Cie HIS20 6 FAM Azide 488 7792 1 EOL CICR EIS 0 5 TAMRA PEG3 Azide 555 7795 1 Edt aus e 5 6 Sulforhodamine 101 PEG3 Azide 7798 1 Eu Se 20 Eterneon Red 645 Azide Cyanine 5 Azide analogue To place your order please contact us under Phone 49 0 721 5606 0 Fax 49 0 721 5606 149 Email info carlroth com 13 ROTH Carl Roth GmbH Co KG Phone 49 0 721 5606 0 Schoemperlenstra2. USER MANUAL TUI EdU Click HTS ROTI kit for High Throughput Screening ROTI kit for High Throughput Screening Carl Roth GmbH Co KG EdU Click HTS ROTI kit for High Throughput Screening 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 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 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 Roth s EdU Click HTS assays overcome these limitations providing a superior alternative to BrdU and 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
3. 6h 4h 2h no EdU 6h EdU incubation time Figure 1 Detection of EdU incorporation depending on cell number and EdU incubation time HeLa cells were seeded in a transparent 96 well cell culture plate with indicated cell numbers per well After 42 h cells were incubated with or without 10 uM EdU for 2 4 or 6 h and subsequently EdU incorporation was detected using the EdU Click HTS 555 Assay Kit and a fluorescence plate reader Mean and SD values from quadruplicates are shown Carl Roth GmbH Co KG ROTI kit for High Throughput Screening 2000 cells 4000 cells 8000 cells 16000 cells 32000 cells 64000 cells gt A2 A10 A11 A12 B11 B12 6 h EdU m m m 4hEdU g m 2hEdU Q no EdU X 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 Well C9 Well G9 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 10 ROTI kit for High Throughput Screening Carl Roth GmbH Co KG Your notes 11 Carl Roth GmbH Co KG ROTI kit for High Throughput Screening Your notes
4. H412 EUH032 P273 ROTI kit for High Throughput Screening Carl Roth GmbH Co KG The rinse buffer Component F is stored at RT and will crystallize at lower temperatures In later case the solution has to be brought to RT mixed thoroughly and can then once homogenously dissolved be used without further considerations The activity of this compound is not affected hereby MSDS the appropriate MSDS can be downloaded from our website www carlroth com Literature Citation When describing a procedure for publication using this product please refer to it as Carl Roth s ROTI kit for High Throughput Screening EdU Click HTS 1 Materials provided with the kit and storage conditions Table 2 Contents of the kit and storage conditions Amount for Amountfor Amount for Component 2 4 20 Vial label Component long term assays well assays well assays well storage storage plates plates plates Component A 5 Ethynyl 2mL 4 mL 20mL 20 C yellow m t m deoxyuridine 5 EdU 6 FAM Azide 5 TAMRA PEG3 Azide 2 8 C Component B 5 6 Sulforhodamine 20 C 1 L 2x1 L 1 L i red a AOE SEIDEL 404 PEG3 Az de dark Dark Eterneon Red 645 Azide Do not Component C freeze 20 mL 40 mL 4x50mL Reaction buffer RT orange Dry Component D 1 mL 1 mL 5 mL Catalyst solution RT green Component E T R blue 200 mg 400 mg 2x1g Buffer additive 20 C Component F girey 6 mL 2x6mL 58mL Rinse buffer 10x RT
5. Click HTS assays are not antibody based and therefore do not require DNA denaturation for detection of the incorporated nucleoside Instead the ROTI kits for High Throughput Screening 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 Carl Roth GmbH Co KG ROTI kit for High Throughput Screening The ROTI kit for High Throughput Screening can be used with antibodies against surface and intracellular markers To ensure the compatibility of your reagent or antibody please refer to Table 1 Table 1 EdU detection dye compatibility Fluorescent molecule Compatibility Organic dyes such as Fluorescein and Compatible Alexa dyes PerCP Allophycocyanin APC and APC Compatible based tandems R phycoerythrin R PE and R PE based Use R PE and R PE based tandems after the tandems 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 Compatibility indicates which involved components are unstable in the presence
6. EEE DE ELITE EN EEE EEE TE IE ET ET DIE EIS i c ag a iy ira ag a ir ducc c Imaging and Analyse Cells At this point the sample can be stored safely 4 Preparation of the stock solutions 4 1 Allow all vials to warm to room temperature before opening 4 1 1 For the preparation of a 20 uM 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 After use store any remaining solution at 20 C When stored as directed this stock solution is stable for up to one year ROTI kit for High Throughput Screening Carl Roth GmbH Co KG Table 3 Amounts of aqueous solution needed to dissolve EdU to a final concentration of 20 uM 1 well plate 1 mL 9 mL 4 1 2 20x EdU solution Component A In dilution Volume for 2x EdU solution in PBS EdU HTS Kit 2 x 96 well plates 2mL 18 mL x 96 well plates 4 mL 36 mL 10 x 96 well plates 10 mL 90 mL 20 x 96 well plates 20 mL 180 mL For the preparation of a stock solution of the buffer additive add the appropriate amount of deionized water see table 4 to the Component E and mix until the compound is dissolved completely After use store 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 Tab
7. 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 This additional wash step with this special rinse buffer reduces unspecific cell number dependent background signal After use store any remaining solution at RT When stored as directed this stock solution is stable for up to 6 months Table 6 Amounts of aqueous solution needed to prepare the 1x rinse buffer Volume of 10x rinse buffer Component F Dilution volume of 1x PBS EdU HTS Kit 1 well plate 2 9 mL 26 1 mL 2 x 96 well plate 5 8 mL 52 2 mL 4 x 96 well plate 11 5 mL 103 5 mL 10 x 96 well plate 28 8 mL 259 2 mL 20 x 96 well plate 57 6 mL 518 4 mL Remove click assay cocktail and wash the cells in each well twice with 150 uL with the 1x rinse solution prepared above 8 5 Remove rinse solution 100 uL of 1 BSA in PBS is then given to the cells in each well 8 6 If performing antibody surface or intracellular labeling proceed immediately to step 9 otherwise continue to step 10 Carl Roth GmbH Co KG ROTI kit for High Throughput Screening 9 Staining intracellular or surface antigens optional 9 1 Add antibodies against intracellular antigens or against surface antigens that use RPE PR tandem or Quantum Dot anti
8. RT This kit is stable up to 1 year after receipt when stored as directed 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 DPBS or TBS Fixative solution 496 Paraformaldehyde in PBS Saponin based permeabilization and wash reagent 10x solution Appropriate cell culture medium 1 BSA bovine serum albumin in PBS pH 7 1 7 4 18 MQ purified water Carl Roth GmbH Co KG ROTI kit for High Throughput Screening 3 Workflow The following protocol was developed using a final EdU concentration of 10 uM and can be adapted for any cell type There are 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 a whole blood sample is used Workflow scheme for the EdU Click HTS Assay Incubate sample with EdU Harvest cells pm HEN AR CM MMC UM Q qer dik d d re d cla MM AMETE Detect Label EdU Wash cells well with rinse buffer qom Se ar ary a a a a a i a a a a a a a eu eee
9. Throughput Screening 5 4 If performing antibody surface labeling proceed immediately to step 6 otherwise continue to step 7 6 Staining cell surface antigens with antibodies optional 6 1 6 2 6 3 6 5 6 6 Wash cells in each well with 100 uL of 1 BSA in PBS Remove the wash solution and add again 100 uL of 1 BSA in PBS to the cells Add surface antibodies and mix well but gently Note PE PE tandem or Quantum Dot antibody conjugates should not be used before performing the click reaction step 8 Incubate the cells for the recommended length of time and temperature Protect from light Proceed to step 7 7 Cell fixation and permeabilization This protocol was developed with a fixation step using 4 Paraformaldehyde in PBS followed by a saponin based permeabilization step The saponin based permeablization and wash reagent can be used with cell samples containing red blood cells or whole blood as well as with cell probes containing different cell types The morphological light scatter characteristics of leukocytes are maintained by the permeabilization reagent while red blood cells are lysed 7 1 7 2 7 3 7 4 Remove the incubation media and wash the cells each well with 100 uL of 1 BSA in PBS Afterwards remove the wash solution Add 100 uL of the fixative solution to the cells in each well Incubate for 15 minutes at room temperature Protect from light Remove the fixation solution and wash the
10. cells in each well twice with 200 uL of 1 BSA in PBS If red blood cells or haemoglobin are present in the sample repeat the washing step Remove all residual blood cell debris and haemoglobin before proceeding NOTE At this point of the procedure the probes can be stored safely Remove the wash solution and add to each well 100 uL of 1x saponin based permeabilization buffer in PBS Mix well but gently and proceed to step 8 for the click reaction 8 EdU detection 8 1 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 Important Once the assay cocktail is prepared use it immediately at least within the next 15 minutes ROTI kit for High Throughput Screening Table 5 Click assay cocktails Carl Roth GmbH Co KG Number of well plates Material Component 2 4 10 Reaction buffer C orange 9 64 mL 19 27 mL 38 54 mL 96 35 mL 192 7 mL Catalyst solution D green 220 uL 440 uL 880 uL 2 2mL 4 4 mL Dye Azide 10 mM B red 55 uL 110 uL 220 uL 550 uL 1 1mL N E blue 11mL 22mL 44mL 11mL 22 mL prepared in 4 1 2 Total Volume 11 01 mL 22 02 mL 44 04 mL 110 1 mL 220 2 mL 8 2 Remove Wash solution from step 7 4 and add 100 uL of the click assay cocktail to each well and mix well but gently to distribute the assay solution evenly 8 3
11. body conjugates Mix well 9 2 Incubate the cells for the time and temperature required for antibody staining Protect from light 9 3 Wash each well twice with 100 uL 1x saponin based permeabilization and wash reagent Remove the solution Add again 100 uL of 1 BSA in PBS to the cells 9 4 Proceed with step 10 for analyzing the cells 10 Imaging and analysis 10 1 Close the 96 well plate by using a sealing film if desired 10 2 Fluorescence is quantified by scanning the plate using an automated imaging platform equipped with filters appropriate for the dye used Images of each well can be taken by microscopy The Excitation and emission maxima of the available dyes are listed in table 7 Table 7 Emission and excitation maxima of the available dyes Product number 7786 1 77871 6 FAM Azide 496 516 Green 7788 1 7789 1 7791 1 5 TAMRA PEG3 Azide 546 579 Violet 7792 1 7793 1 77941 5 6 Sulforhodamine 101 PEG3 584 603 Grang 7795 1 fiue TB Eterneon Red 645 Azide 7797 1 643 662 Red 7798 1 Cyanine 5 Azide analogue Excitation nm Emission nm Filter ROTI kit for High Throughput Screening Carl Roth GmbH Co KG 11 Example of the data derived from an EdU Click HTS Kit based experiment Proliferation of HeLa cells 50000 Cell v L number 2 40000 ulated 9 i 2000 30000 ili 4000 9 ii 8000 i4 16000 20000 14 32000 z 14 64000 F 10000 0 sch
12. e 3 5 Fax 49 0 721 5606 149 76185 Karlsruhe Germany Email info carlroth com
13. le 4 Amounts of aqueous solution needed to dissolve the buffer additive to the final work solution 10 x 96 well plates 1g 10 mL 20 x 96 well plates 2g 25 mL Buffer additive solid Component E Dilution volume of deionized water EdU HTS Kit 1 well plate 100 mg 1 mL 2 x 96 well plates 200 mg 2 5 mL 4 x 96 well plates 400 mg 5 mL 5 Labeling of cells with EdU 5 1 5 2 5 3 Suspend the cells in an appropriate tissue culture medium to obtain optimal cell growth conditions Please note that the growth of the cells during incubation decelerates if the temperature changes or the cells are washed prior to incubation with EdU 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 uM for 1 4 hours as a starting point Use higher EdU concentrations for a shorter incubation time A longer incubation time requires lower EdU concentrations The incubation of the cells with EdU should be performed under the optimal conditions for your cell type the number of cells plated and for the desired length of time Various DNA synthesis and proliferation parameters can be evaluated by altering the EdU incubation time or by subjecting the cells to pulse labeling with EdU Effective time intervals for pulse labeling and the length of each pulse depend on the cell growth rate Carl Roth GmbH Co KG ROTI kit for High
14. of copper catalyst for the EdU detection reaction either the fluorescent dye itself or the detection method Not all GFP antibodies recognize the same antigen site Rabbit and chicken anti GFP antibodies result in a good fluorescent amount The mouse monoclonal antibodies tested are not recommended for this application because they do not generate an acceptable amount of fluorescence For research use only Information in this document is subject to change without notice Carl Roth GmbH Co KG assumes no responsibility for any errors that may appear in this document Carl Roth GmbH Co KG 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 Carl Roth GmbH Co KG 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 this kit Cautions EdU Component A amp Danger H340 H360 P202 P280 P308 P313 Reaction Buffer Component C lt gt Warning H315 H319 P280 P302 P352a P305 P351 P338 Catalyst Solution Component D amp lt gt Warning H302 H315 H319 H400 H410 P280 P301 P312a P302 P352a P305 P351 P338 Rinse Buffer Component F
Download Pdf Manuals
Related Search