EdU Flow Cytometry Kit User Manual - Sigma
Contents
1. 1 Materials provided with the Kit and storage conditions Table 2 Contents of the kit and storage conditions Component Amount for Amount for Vial label Component long term Kit storage 50 assays 100 assays storage 6 FAM Azide BCK FC488 5 TAMRA PEG3 Azide BCK FC555 a 130 uL 2x130uL 5 6 Sulforhodamine101 PEG3 Azide a BCK FC594 C 5 Azide BCK FC647 Fe zide gt ge Dark Dry Fixative solution 4 L 2 L Do not freeze Component E 50 mL 3 x50 mL Saponin based permeabilization and wash reagent 10x solution green 400 mg 400 mg Buffer additive This kit is stable up to 1 year after receipt when stored as directed 2 Required Material and Equipment not included in this Kit e Adherent cells e Reaction tubes size depends on the volume of reaction cocktail needed e Buffered saline solution such as PBS D PBS or TBS e Appropriate cell culture medium e 1 BSA bovine serum albumin in PBS pH 7 1 7 4 e Deionized water or 18 MQ purified water e Flow cytometry tubes 3 Workflow The following protocol was developed using an 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 EdU Flow Cytometry Kit ba Seclick2. baseclick EdU Flow Cytometry Kit User Manual EdU Flow Cytometry Kit baseclick Ordering information for detailed kit content see Table 2 EdU Flow Cytometry Kits for 50 assays BCK FC488 50 FAM Azide BCK FC555 50 5 TAMRA PEG3 Azide BCK FC594 50 5 6 Sulforhodamine 101 PEG3 Azide BCK FC647 50 Cyanine 5 Azide EdU Flow Cytometry Kits for 100 assays BCK FCA88 100 20mg SFAM Azide BCK FC555 100 20mg 5 TAMRA PEG3 Azide BCK FC594 100 20mg 5 6 Sulforhodamine 101 PEG3 Azide BCK FC647 100 20mg Cyanine 5 Azide To place your order please contact us under e phone 49 8158 903867 e fax 49 8158 903894 e email orders baseclick eu baseclick EdU Flow Cytometry Kit EdU Flow Cytometry Kit 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
3. 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 Flow Cytometry Assay Incubate sample with EdU Analyse cells by flow cytometry 4 Preparation of the stock solutions 4 1 Allow all vials to warm to room temperature before opening 4 2 For the preparation of a 10 mM stock solution of EdU add the appropriate amount of DMSO component C or aqueous solution 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 Table 3 Amounts of DMSO or aqueous solution needed to dissolve EdU to a final concentration of 10 mM EdU Flow Cytometry Kit EdU amount DMSO aqueous solution amount 4 3 For the preparation of a 10x stock solution of the buffer additive add 4 mL of deionized water to the component G 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 If the solution starts to EdU Flow Cytometry Kit 4 4 baseclick develop a brown colour it has degraded and should be discarded We recommend preparing aliquots to avoid repeated thaw and freeze cycles For the preparation of 500 mL of the
4. 1x saponin based permeabilization buffer and wash reagent for 50 assays add 50 mL of component E to 450 mL of 1 BSA in PBS For the preparation of 1 L of the 1x saponin based permeabilization buffer and wash reagent for 100 assays add 100 mL of component E to 900 mL of 1 BSA in PBS After use store any remaining solution at 2 8 C Note The saponin based permeabilization and wash reagent contains sodium azide Please see the cautions on page 4 5 Labeling of cells with EdU 5 1 5 2 5 3 5 4 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 2 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 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
5. growth rate Harvest cells 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 4 6 5 6 6 Wash cells with 3 mL of 1 BSA in PBS Centrifuge to pellet cells and remove supernatant Dislodge the pellet and resuspend cells in 1 BSA in PBS at 1 x 10 cells mL Add 100 uL of cell suspension or whole blood sample to flow tubes Add surface antibodies and mix well 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 EdU Flow Cytometry Kit ba Seclick 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 suspensions containing red blood cells or whole blood as well as with cell suspensions 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 Remove the incubation media and wash the cells with 3 mL of 1 BSA in PBS Pellet the cells and remove the supernatant 7 2 Dislodge the cell pellet Add 100 uL of
6. 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 baseclick 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 Cautions DMSO Component C is known to facilitate the entry of organic molecules into tissues DMSO is irritant and flammable Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials Dispose of the reagents in compliance with all related local arrangements Fixative solution Component D contains paraformaldehyde which is harmful Use with appropriate precautions Saponin based permeabilization and wash reagent Component E contains sodium azide which is highly toxic and yields the extremely toxic hydrazoic acid under acidic conditions Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumping This solution is orange EdU Flow Cytometry Kit base click Literature Citation When describing a procedure for publication using this product please refer to it as baseclick EdU Flow Cytometry Kit
7. Kit ba Seclick 8 4 Wash the cells with 3 mL of 1x saponin based permeabilization and wash reagent prepared in 4 4 Pellet the cells and remove the supernatant Dislodge the cell pellet If proceeding with intracellular antibody labeling in step 9 resuspend the cells in 100 uL of 1x saponin based permeabilization and wash reagent Otherwise add 500 uL of 1x saponin based permeabilization and wash reagent and proceed with step 10 for analyzing the cells with a flow cytometer Important Keep the samples protected from light during the whole procedure 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 antibody conjugates Mix well 9 2 Incubate the tubes for the time and temperature required for antibody staining Protect from light 9 3 Wash each tube with 3 mL 1x saponin based permeabilization and wash reagent prepared in 4 4 Pellet the cells and remove the supernatant Dislodge the cell pellet and resuspend the cells in 500 uL of 1x saponin based permeabilization and wash reagent 9 4 Proceed with step 10 for analyzing the cells with a flow cytometer 10 Imaging and analysis Use a low flow rate during acquisition if a traditional flow cytometer with a hydrodynamic focusing is used to measure the total DNA content The same collection rate and cell concentration should be used for each sample within an e
8. low Cytometry Kit baseclick The baseclick EdU Flow Cytometry Kit 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 EdU tandems 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 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 baseclick GmbH assumes no responsibility for any errors that may appear in this document baseclick GmbH disclaims all warranties with
9. rs The click reaction using 6 FAM Azide was performed according to the recommended staining protocol Fluorescence intensity of 10 000 cells was measured by flow cytometry The results are presented in form of histograms showing the cell number in the y axis and the FL1 Fluorescence in the x axis FL1 voltage setting was adjusted according to the fluorescence signal of the negative cell population 333 V with 6 FAM 1A represents the negative control of proliferating and non proliferating cells without EdU incorporation 1B shows non proliferating cells without EdU incorporation left peak and proliferating cells S phase which have incorporated EdU and are labelled with 6 FAM Azide right peak The baseclick EdU Flow Cytometry Kit is compatible with several cell cycle dyes An example using 6 FAM Azide is illustrated in Figure 2 Un 2A 2B FL1 positive L FL1 positive 4 4 aa u eh FL1 negative u 4 FL1 negative G1 S G2 M G1 S G2 M FL3 A azi FL3 A nu Figure 2 Density blots of Propidium lodide PI stained samples Samples of HeLa cells treated without 2A or with EdU 2B were incubated with 10 uM EdU for 2 hours The reaction cocktail carrying 6 FAM Azide was used After the click reaction DNA was stained using PI FL3 fluorescent channel The y axis presents the FL1 Fluorescence intensity and the x axis the content of DNA measured with FL3 area Cell cycle phases are indicated as G1 S and G2 M phase EdU F
10. 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 baseclick EdU Flow Cytometry Kits overcome these limitations providing a superior alternative to BrdU and H thymidine assays for directly measuring DNA synthesis 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 Flow Cytometry Assays are not antibody based and therefore do not require DNA denaturation for detection of the incorporated nucleoside Instead the EdU Flow Cytometry Kits 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 EdU Flow Cytometry Kit Sbaseciick Standard flow cytometry methods are used to determine the percentage of S phase cells in the population Figure 1 1A 136 a JJ FL1 Height BR FL1 Height On Figure 1 Fluorescence histograms of EdU incorporation with baseclick EdU Flow Cytometry Kit Samples of HeLa cells treated without 1A or with EdU 1B were incubated with 10 uM EdU for 2 hou
11. the fixative solution component D to the cells Mix well and incubate for 15 minutes at room temperature Protect from light 7 3 Remove the fixation solution and wash the cells with 3 mL of 1 BSA in PBS Pellet the cells and remove the supernatant 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 7 4 Dislodge the cell pellet Resuspend the cells in 100 uL of 1x saponin based permeabilization buffer in PBS prepared in 4 4 Mix well and proceed to step 8 for the click reaction 8 EdU detection 8 1 Prepare the assay cocktail in the same order as described in table 4 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 Table 4 Click assay cocktails Number of assays Material Component 1 2 3 5 10 PBS D PBS or TBS met 438 uL 875 uL 1 32mL 2 19mL 4 38 mL provided Catalyst solution 10 uL 20 uL 30 uL 50 uL 100 uL Buffer additive 10x 50 uL 100 uL 150 uL 250 uL 500 uL prepared in 4 3 6 som 100p sou 250m soont 8 2 Add the appropriate amount of the assay cocktail to the cells and mix well to distribute the assay solution evenly 8 3 Incubate the assay mixture for 30 minutes at room temperature Protect from light EdU Flow Cytometry
12. xperiment Detect the fluorescent signal generated by DNA content stains with linear amplification The fluorescent signal generated by EdU labeling is best detected with logarithmic amplification The Excitation and emission maxima of the available dyes are listed in table 5 Table 5 Emission and excitation maxima of the available dyes BCK FC555 5 TAMRA PEG3 Azide Product number Dye Excitation nm Emission nm Filter e 5 6 Sulforhodamine 101 me EEE BCK FC647 Cyanine 5 Azide baseclick EdU Flow Cytometry Kit Your notes 10 EdU Flow Cytometry Kit User Manual Version 2 6 baseclick baseclick GmbH Phone 49 8158 903867 Bahnhofstra e 9 15 Fax 49 8158 903894 82327 Tutzing Germany Email info baseclick eu
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