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1. basecliok EdU in vivo Kits BCK IV This kit contains chemicals to perform up to 400 reactions depending on your purchased kit You purchased the following EdU in vivo Kit Here you will find the same stick on label The precedent User Manual is completed attached on the kit you purchased by a second User Manual which complies with the ordered specific kit EdU in vivo Kit baseclick Ordering information EdU in vivo Imaging Kits Kit code EdU Combined with Dye BCK555 IV IM S 50 mg EdU Click Kit 5 TAMRA PEG3 Azide BCK594 IV IM S 50 mg EdU Click Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK594 IV IM M 500 mg EdU Click Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK488 IV IM L 1000 mg EdU Click Kit 6 FAM Azide BCK594 IV IM L 1000 mg EdU Click Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK647 IV IM L 1000 mg EdU Click Kit Cyanine 5 Azide Other Kits Amounts and dyes upon request EdU in vivo Flow Cytometry Kits Kit code EdU Combined with Dye BCK555 IV FC S 50 mg EdU Flow Cytometry Kit 5 TAMRA PEG3 Azide BCK594 IV FC S 50 mg EdU Flow Cytometry Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK647 IV FC S 50 mg EdU Flow Cytometry Kit Cyanine 5 Azide BCK IV FC M BCK488 IV FC M 500 mg EdU Flow Cytometry Kit 6 FAM Azide BCK555 IV FC M 500 mg EdU Flow Cytometry Kit 5 TAMRA PEG3 Azide BCK594 IV FC M 500 mg EdU Flow Cytometry Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK647 IV FC M 500 mg EdU Flow Cytometry Kit Cyanine 5 A
2. EdU in the appropriate amount of phosphate buffered saline PBS according to table 2 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 one year Edu Amountof PBS 50 mg 20 mL 100 mg 40 mL 1g 400 mL 5g 2000 mL Table 2 Amounts of PBS needed to dissolve EdU for a final concentration of 10 mM The preparation of other stock solutions are described in the according User Manual you get with your kit or can be downloaded from our website www baseclick eu EdU in vivo Kit ba Seclick 4 EdU Administration We recommend testing a range of EdU concentrations to determine the optimal concentration for your experiment If currently using BrdU based assays a similar concentration and incubation time to BrdU is a good starting point for EdU The optimal concentration may vary depending upon the duration of the pulse Lower concentrations are recommended for longer incubation time General recommendations are listed below See point 5 EdU administration to the animals can be performed following injection or media incorporation Successful EdU labeling in animals has been reported for the following species Mouse Salic A et al Proc Natl Acad Sci USA 2008 105 2415 2420 Rat Guo J et al Cardiovascular Diabetology 2012 11 150 Nematode C elegans Dorsett M Westlund B Schedl T Genetics 2
3. e Allow slides containing mounted frozen tissue sections to thaw and warm to room temperature e Fix tissue sections with 4 Paraformaldehyde in PBS pH 7 4 for 15 minutes e Wash twice with 3 BSA in PBS e Permeabilize the tissue sections with 0 5 Triton X 100 in PBS for 20 minutes e Wash twice with 3 BSA in PBS EdU detection see step 7 Chicken Gallus domesticus Reference Kaiser CL Kamien AJ Shah PA Laryngoscope 2009 119 1770 1775 EdU administration e Perform a single subcutaneous injection of EdU 50 mg kg in sterile PBS pH 7 4 e Euthanize the bird 8 hours after EdU injection e Remove the tissue organ e Snap freeze the tissue organ e Section the tissue organ at 20 um using a cryostat and mount onto appropriate Slides Cell fixation and permeabilization For flow cytometry analysis see step 6 For imaging analysis e Fix the cells in chilled 4 Paraformaldehyde in PBS for 1 hour e Rinse 3 times in PBS for 5 minutes each rinse EdU detection see step 7 EdU in vivo Kit ba Seclick 6 Cell fixation and permeabilization for flow cytometry analysis See also according user User Manual 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 ce
4. way design your experiment individually If you wish to purchase another dye or azide component please contact us for price inquiry EdU in vivo Kit ba seclick Generally following standard amounts of EdU are delivered with the kits In order to freely choose the right kit for your special in vivo application our in vivo kits are designed in a sense that you combine one of our three baseclick EdU cell proliferation kits such as Imaging kit BCK IV IM flow cytometry kit BCK IV FC and our high throughput screening HTS kit BCK IV HTS with the dye of choice and the right EdU content for your animal model Depending on your animal or the number of animals to be tested you can choose between three kit sizes S M and L with increasing EdU content as indicated in the following table Kit size Content of EdU 50 mg M 500 mg 1000 mg Other EdU amounts available upon request To place your order please contact us under e phone 49 8158 903867 e fax 49 8158 903894 e webshop www baseclick eu e email orders baseclick eu EdU in vivo Kit baseclick EdU in vivo 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 inco
5. 009 183 233 247 Cricket Gryllus bimaculatus Bando T Mito T Maeda Y et al Development 2009 136 2235 2245 Chicken Gallus domesticus Kaiser CL Kamien AJ Shah PA Laryngoscope 2009 119 1770 1775 Zebra Fish larva Danio rerio Luedtke W et al PNAS 2011 108 51 5 General recommendations for different animals Mouse Rat EdU can be injected in different doses from 10 to 200 mg kg Using 50 mg kg of EdU in PBS i p results in near maximal intensity of proliferated cells Reference for mouse Salic A et al Proc Natl Acad Sci USA 2008 105 2415 2420 Reference for rat Guo J et al Cardiovascular Diabetology 2012 11 150 EdU administration e We recommend to perform a single intraperitoneal injection of EdU in PBS 50 mg kg e Euthanize the mouse 4 hours after EdU injection e Remove the desired tissue organ e Snap freeze the tissue organ e Section the tissue organ at 20 um using a cryostat and mount onto appropriate Slides Cell fixation and permeabilization For flow cytometry analysis see step 6 For imaging analysis e Allow slides containing mounted frozen tissue sections to thaw and warm to room temperature e Fix tissue sections with 4 Paraformaldehyde in PBS pH 7 4 for 15 minutes EdU in vivo Kit ba Seclick e Wash twice with 3 BSA in PBS pH 7 4 e Permeabilize the tissue sections with 0 5 Triton X 100 in PBS for 20 minutes e Wash twice with 3 BSA in PBS EdU detecti
6. ance to perform in vivo studies Contact your local authorities for the animal testing regulations and authorization of your in vivo experiments if not already done Cautions EdU is a nucleoside analogue which can be incorporated into DNA Handle and dispose EdU in compliance with all pertaining local regulations EdU is soluble in DMSO water alcohol and aqueous buffers For in vivo experiments EdU can be solved in a buffered saline solution such as PBS EdU toxicity No cytotoxicity could be measured up to 1mM EdU The data on the pharmacotoxicity of EdU are in publication process at the moment by baseclick Fixative solution BCK IV FC S M amp L contains paraformaldehyde which is harmful Use with appropriate precautions Saponin based permeabilization and wash reagent BCK IV FC S M amp L 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 Rinse buffer BCK IV HTS S M amp L contains hazardous components Use with appropriate precautions Keep away from acids to avoid dangerous gases D sande reagents containing the rinse buffer using equipment and practices appropriate for the hazards posed by such materials Use gloves Dispose of the reagents in compliance with all related local arrangements For the c
7. d to analyze the distribution of EdU in different organs 0 00 Psi 5 25 5 25 50 EdU Dose mg kg EdU Dose mg kg Lymphnode 4 00 3 300 5 sa 2 2 00 sa 3 A Region 3 100 0 00 5 25 EdU Dose mg kg EdU Dose mg kg The shown in vivo experiments were performed in collaboration with a third party after having received the allowance of the local authorities 11 EdU in vivo Kit ba seclick Your notes EdU in vivo Kit User Manual Version 3 1 baseclick baseclick GmbH Phone 49 8158 903867 Bahnhofstra e 9 15 Fax 49 8158 903894 82327 Tutzing Germany Email info baseclick eu 13
8. ll types The morphological light scatter characteristics of leukocytes are maintained by the permeabilization reagent while red blood cells are lysed 6 1 Wash the cells with 3 mL of 1 BSA in PBS Pellet the cells and remove the supernatant 6 2 Dislodge the cell pellet Add 100 uL of the fixative solution to the cells Mix well and incubate for 15 minutes at room temperature Protect from light 6 3 Remove the fixation solution and wash the cells with 3 mL of 1 BSA in PBS Pellet cells and remove the supernatant Remove all residual blood cell debris and haemoglobin before proceeding 6 4 Dislodge the cell pellet Resuspend the cells in 100 ul of 1x saponin based permeabilization buffer in PBS Mix well and proceed to step 7 for the click reaction 7 EdU detection See according User Manuals 8 Imaging flow cytometry analysis See according User Manuals 10 EdU in vivo Kit baseclick 9 Example of the data derived from an EdU in vivo Kit based experiment This EdU in vivo kit allows detection of cell proliferation in whole animals showing no toxic effects Cell proliferation is detected in different organs by applying the reagents of this kit In healthy mice cell proliferation is nicely detected in lymphnodes but not in other organs showing the reliability of this baseclick method Table 7 NMRI mice treated with different concentrations of EdU intraperitoneally and sacrificed after 4hrs A flow cytometric method is use
9. on see step 7 Nematode C elegans Reference Dorsett M Westlund B Schedl T Genetics 2009 183 233 247 EdU administration e Prepare EdU plates grow MG1693 Escherichia coli thymidine deficient overnight at 37 C Dilute the cells 1 50 in 1 glucose 1 25 ug mL thiamine 0 5 uM thymidine 1 mM MgSO and 20 uM EdU in M9 minimal media Grow this culture at 37 C for 24 hours in the dark harvest bacteria and resuspend in a small volume of M9 minimal media Plate the suspension on 60 mm M9 plates e For labelingproliferative cells place the worms into the EdU plates for 3 hours e Wash the plates in PBS to collect the worms and dissect them Cell fixation and permeabilization For flow cytometry analysis see step 6 For imaging analysis e Fix sections in 3 Paraformaldehyde in 0 1 M K HPO pH 7 2 for 5 minutes at room temperature EdU detection see step 7 Cricket Gryllus bimaculatus Reference Bando T Mito T Maeda Y et al Development 2009 136 2235 2245 EdU administration e We recommend to perform a single intraperitoneal injection of EdU in PBS 50 mg kg e Euthanize the cricket 4 hours after EdU injection e Remove the desired tissue organ e Snap freeze the tissue organ e Section the tissue organ at 20 um using a cryostat and mount onto appropriate slides EdU in vivo Kit ba Se lick Cell fixation and permeabilization For flow cytometry analysis see step 6 For imaging analysis
10. orrect handling we refer you to the MSDS which can be downloaded from our webpage www baseclick eu MSDS the appropriate MSDS for all components of each individual kit can be downloaded from our website www baseclick eu Literature Citation When describing a procedure for publication using this product please cite it as the EdU in vivo Kit from baseclick GmbH EdU in vivo Kit baseclick 1 Material provided and storage conditions For the specification of the material provided and their storage conditions please see the specific user manual of your purchased kit which is included in your baseclick box or can be downloaded from our webpage since each kits has its proper composition 2 Required Material and Equipment The required materials and equipment depends on your provided kit please see the specific kit s user manual delivered with the kit you purchased e Reaction tubes size depends on the volume of reaction cocktail needed e Buffered saline solution such as PBS D PBS or TBS e Fixation solution 3 4 Paraformaldehyde in PBS e Permeabilization solution for example 0 5 Triton X 100 in PBS e 1 BSA bovine serum albumin in PBS pH 7 1 7 4 e Deionized water or 18 MO purified water already included in flow cytometry kits BCK IV FC S M amp L 3 Preparation of the EdU stock solution 3 1 Allow all vials to warm to room temperature before opening 3 2 For the preparation of 10 mM EdU solution dissolve
11. rich results The baseclick EdU in vivo Kit is compatible with several cell cycle dyes and 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 Fluorescent proteins e g GFP Use anti GFP antibodies before the EdU detection reaction or use organic dye based reagents for protein expression detection EdU in vivo Kit ba Seclick 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 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 This is not an allow
12. rporation 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 The baseclick EdU in vivo Kits overcome these limitations providing a superior alternative to BrdU and H thymidine assays for directly measuring DNA synthesis in vivo 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 in vivo Kits are not antibody based and therefore do not require DNA denaturation for detection of the incorporated nucleoside Instead the EdU in vivo Kits utilize a fast efficient and reliable method based on the so called 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
13. zide BCK488 IV FC L 1000 mg EdU Flow Cytometry Kit 6 FAM Azide BCK594 IV FC L 1000 mg EdU Flow Cytometry Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK647 IV FC L 1000 mg EdU Flow Cytometry Kit Cyanine 5 Azide Other Kits Amounts and dyes upon request EdU in vivo Kit baseclick EdU in vivo High Throughput Screening HTS Kits Kit code EdU Combined with Dye BCK594 IV HTS S 50 mg EdU HTS 2x96 Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK555 IV HTS M 500 mg EdU HTS 2x96 Kit 5 TAMRA PEG3 Azide BCK594 IV HTS M 500 mg EdU HTS 2x96 Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK488 IV HTS L 1000 mg EdU HTS 2x96 Kit 6 FAM Azide BCK555 IV HTS L 1000 mg EdU HTS 2x96 Kit 5 TAMRA PEG3 Azide BCK594 IV HTS L 1000 mg EdU HTS 2x96 Kit 5 6 Sulforhodamine 101 PEG3 Azide BCK647 IV HTS L 1000 mg EdU HTS 2x96 Kit Cyanine 5 Azide Other Kits Amounts and dyes upon request Generally following standard dyes are delivered with the kits Absorption emission E Analogous dyes Aabs 496 Aem 516 83000 cm M Alexa 488 DyLight488 CFAM Azide FluorX ATTO488 5 TAMRA PEG3 Aabs 546 Aem 579 91000 cm 1M 1 Alexa 555 Cy3 Azide 5 6 Sulforhodamine Aaps 584 Aem 603 116000 cm M Texas Red Alexa 594 101 PEG3 Azide ATTO 594 Xabs 646 Aem 662 250000 cm M Alexa 647 Cy5 Cyanine 5 Azide ATTO647 For each kit you can choose between all our azide terminated dyes on www baseclick eu and in that

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