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OxiSelect™ Cellular UV-Induced DNA Damage

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1. Product Manual OxiSelect Cellular UV Induced DNA Damage Staining Kit CPD Catalog Number STA 327 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Absorption of ultraviolet UV light produces two predominant types of DNA damage cyclobutane pyrimidine dimers CPD and pyrimidine 6 4 pyrimidone photoproducts 6 4PP Figure 1 The result is a transition of C to T and CC to TT which are the most frequent mutations of p53 in both human and mouse skin cancers UV damaged DNA is usually repaired by nucleotide excision repair NER or base excision repair BER After UV exposure cells activate p53 and stall the cell cycle for repair If the damage is too severe the cell will trigger apoptosis to get rid of DNA damaged potentially mutant cells Cell Biolabs OxiSelect Cellular UV induced DNA Damage Staining Kit CPD is an immunofluorescence assay developed for rapid detection of CPDs in genomic DNA of cultured cells Each kit provides sufficient reagents for up to 96 stainings in a 96 well plate 8 0 R R HN S nn 9 Ant tuo R r 1 13 0 on aw CPD 5 ye dipyrimidines 6 4 photoproduct Figure 1 Structures of DNA lesions induced by UV Light Assay Principle Cells are first seeded in a 96 well tissue culture plate Wells are then UV irradiated to induce DNA damage After fixation and denaturatio
2. other plate formats will decrease the number of assays possible with this kit Culture Dish 96 well 48 well 24 well 12 well 6 well DPBS during UV Irradiation uL well oe ay u goo tono 75 Methanol 25 Acetic Acid 100 200 400 800 1600 uL well 70 Ethanol uL well 100 200 400 800 1600 Denaturation Solution A 100 200 400 800 1600 uL well DEBS dunne Washing 206 400 800 1600 3200 uL well Denaturation Solution B 100 200 400 800 1600 uL well asray Datuept Blockne 566 400 800 1600 3200 uL well 1X Anti CPD Antibody Solution ul veil 100 200 400 800 1600 1X Secondary Antibody FITC Conjugate Solution 100 200 400 800 1600 uL well Wash Buffer uL well 250 500 1000 2000 4000 Table 1 Dispensing Volumes of Different Plate Formats 4 AN 5 CELL BIOLABS INC JE a I Cell Seeding 1 Harvest and resuspend cells in culture medium at 2 4 x 10 cells mL Seed 100 uL in each well of a 96 well tissue culture plate and incubate overnight at 37 C 5 CO cells should be gt 80 confluent II UV Treatment Fixation and Denaturation 1 Carefully remove medium from the wells by tilting the plate and aspirating from the edge Gently add 100 uL of DPBS containing magnesium and calcium to each well taking care not to dislodge the cells Perform UV irradiation to desired wells removal of plate cover is recommended Include wells without irradiati
3. 3 510 514 4 Soehnge H Ouhtit A Ananthaswamy ON 1997 Front Biosci 2 D538 D551 el Deiry WS Tokino T Velculescu VE Levy DB Parsons R Trent JM Lin D Mercer WE Kinzler KW Vogelstein B 1993 Cell 75 817 825 6 Hermeking H Lengauer C Polyak K He TC Zhang L Thiagalingam S Kinzler KW Vogelstein B 1997 Mol Cell 1 3 11 7 Hill LL Ouhtit A Loughlin SM Kripke ML Ananthaswamy HN Owen Schaub LB 1999 Science 285 898 900 Recent Product Citation N ez Lozano R et al 2015 Biocompatible films with tailored spectral response for prevention of DNA damage in skin cells Adv Healthc Mater doi 10 1002 adhm 201500223 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolab
4. Diluent Part No 310804 One 50 mL bottle 10X Wash Buffer Part No 250007 One 50 mL bottle Materials Not Supplied Se eS a e ee eS 96 well tissue culture plate Cell line of interest UV crosslinker irradiator or germicidal lamp DPBS containing magnesium and calcium 75 Methanol 25 Acetic Acid 70 Ethanol 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Fluorescence microscope with FITC filter 3 JaN CELL BIOLABS INC A a Storage Store all kit components at 4 C until their expiration dates Preparation of Reagents e 1X Wash Buffer Dilute the 10X Wash Buffer Concentrate to 1X with deionized water Stir to homogeneity e Anti CPD Antibody and Secondary Antibody FITC Conjugate Immediately before use dilute the Anti CPD Antibody 1 100 and Secondary Antibody 1 100 with Assay Diluent Do not store diluted solutions e Denaturation Solution A Immediately before use dilute the Denaturation Solution A 1 100 with 70 Ethanol Do not store diluted solution e Denaturation Solution B Immediately before use dilute the Denaturation Solution B 1 100 with DPBS containing magnesium and calcium Do not store diluted solution Assay Protocol The following assay protocol is written for a 96 well format Refer to the below table for the appropriate dispensing volumes of other plate formats Note using
5. d immediately to the next step 5 Add 100 uL of DPBS to each well View staining with a fluorescence microscope using FITC filter 5 JaN CELL BIOLABS INC A a Example of Results The following figures demonstrate typical Cellular UV induced DNA Damage Staining Kit CPD results One should use the data below for reference only This data should not be used to interpret actual results Secondary UValireated Nn tibodygAlone Figure 2 DNA Damage Induced by UV Light in HeLa Cells HeLa cells were seeded at 20K well overnight then exposed to light under a germicidal lamp for 30 minutes Immunofluorescence staining of CPD damage was determined as described in the Assay Instructions 1 6 1 4 1 2 1 0 0 8 0 6 0 4 0 2 0 0 Secondary UV Treated Untreated Antibody Alone OD 450nm Figure 3 DNA Damage Induced by UV Light in HeLa Cells HeLa cells were seeded at 20K well overnight then exposed to light under a germicidal lamp for 30 minutes Relative CPD damage was determined using the OxiSelect Cellular UV induced DNA Damage ELISA Kit STA 326 6 CELL BIOLABS INC es References 1 Lippke JA Gordon LK Brash DE Haseltine WA 1981 Proc Natl Acad Sci U S A 78 3388 3392 2 Mitchell DL Nairn RS 1989 Photochem Photobiol 49 805 819 Ananthaswamy HN Loughlin SM Cox P Evans RL Ullrich SE Kripke ML 1997 Nat Med
6. n cells containing CPD damage are probed with an anti CPD antibody followed by a FITC conjugated secondary antibody The unbound secondary antibody is removed during a wash step and stained cells can then be visualized with a fluorescence microscope 2 JA CELL BIOLABS INC AN Related Products SO Oy eS SNS STA 320 OxiSelect Oxidative DNA Damage ELISA Kit 8 OHdG Quantitation STA 321 OxiSelect DNA Double Strand Break DSB Staining Kit STA 322 OxiSelect UV induced DNA Damage ELISA Kit CPD Quantitation STA 323 OxiSelect UV induced DNA Damage ELISA Kit 6 4PP Quantitation STA 324 OxiSelect Oxidative DNA Damage Quantitation Kit AP sites STA 325 OxiSelect Oxidative RNA Damage ELISA Kit 8 OHG Quantitation STA 326 OxiSelect Cellular UV induced DNA Damage ELISA Kit CPD STA 328 OxiSelect Cellular UV induced DNA Damage ELISA Kit 6 4PP STA 329 OxiSelect Cellular UV induced DNA Damage Staining Kit 6 4PP 10 STA 351 OxiSelect Comet Assay Kit 3 Well Slides 75 Assays 11 STA 353 OxiSelect Comet Assay Slides 3 Well 25 Slides 12 STA 355 OxiSelect 96 Well Comet Assay Kit Kit Components Ne a Oe SS Anti CPD Antibody 100X Part No 232601 One 100 uL vial Secondary Antibody FITC Conjugate 100X Part No 232102 One 100 uL amber vial Denaturation Solution A 100X Part No 232602 One 200 uL vial Denaturation Solution B 100X Part No 232603 One 200 uL vial Assay
7. on as a negative control Samples should be performed in triplicate Aspirate the wells and add 100 uL of 75 Methanol 25 Acetic Acid to each well Incubate 30 minutes at room temperature Aspirate the wells and add 100 uL of 70 Ethanol to each well Incubate 30 minutes at room temperature Aspirate the wells and add 100 uL of Denaturation Solution A see Preparation of Reagents to each well Incubate 5 minutes at room temperature Gently wash 3 times with 200 uL DPBS containing magnesium and calcium 7 Aspirate the wells and add 100 uL of Denaturation Solution B see Preparation of Reagents to each well Incubate 10 minutes at room temperature Aspirate the wells and add 200 uL of Assay Diluent to each well Block the wells 30 minutes at room temperature II CPD Immunofluorescence Staining 1 Aspirate the wells and add 100 uL of the diluted anti CPD antibody see Preparation of Reagents to each well Incubate at room temperature for 1 hour on an orbital shaker Wash microwell strips 4 times with 250 uL 1X Wash Buffer per well with thorough aspiration between each wash After the last wash empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer Add 100 uL of the diluted Secondary Antibody FITC Conjugate see Preparation of Reagents to each well Incubate at room temperature for 1 hour on an orbital shaker Wash microwell strips 4 times according to step 2 above Procee
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