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1. Pry Cellular UV DNA Damage Detection Kit od ycLex User s Manual MBI For Research Use Only Not for use in diagnostic procedures ernational Corporation Cell Based ELISA Kit for Measuring cyclobutane pyrimidine dimers in situ CycLex Cellular UV DNA Damage Detection Kit Cat CY 1141 For 200 Assays Intended UW l AA IIIA l Introduction 2 Principle of the Assay 2 4 Materials Provided 5 Materials Required but not Provided 5 Precautions and Recommendation 6 Detailed Protocol 7 10 Troubleshooting w w wowamoa 10 Reagent Stability 10 Example of Test ResultS 11 14 RAS Wa 15 Related ProductS vs 15 Intended Use The CycLex Research Product CycLex Cellular UV DNA Damage Detection Kit is a non isotopic immunoassay used for the semi quantitative measurement of CPDs cyclobutane pyrimidine dimers in genomic DNA formed by UV irradiation to cells Applications for this kit include 1 Detection and semi quantification of cyclobutane pyrimidine dimers in cells 2 Monitoring the effects of UV on formation of cyclobutane pyrimidine dimers in cells 3 Monitoring the effects of UV protection reagent on formation of2. For Research Use Only Not for use in diagnostic procedures mernational Corporation wy YCLEX Fig 4 Effect of cell number on ELISA value using Raji cells CPDs Raji e UVO O UV 0 156J m2 a UV 0 625J m2 UV 2 5J m2 A UV 10J m2 A UV 40J m2 12 500 25 000 50 000 Cell number Cat CY 1141 14 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit y cd cLex User s Manual pa BL y For Research Use Only Not for use in diagnostic procedures mernational Corporation References Nishiwaki Y et al J Invest Dermatol 122 526 532 2004 Imoto K et al J Invest Dermatol 119 1177 7782 2002 Kobayashi N et al Pigment Cell Res 14 94 102 2001 Katsumi S et al J Invest Dermatol 117 1156 1161 2001 Otoshi E et al Cancer Res 60 1729 1735 2000 Nakagawa A et al J Invest Dermatol 110 143 148 1998 Kobayashi N et al J Invest Dermatol 110 806 810 1998 Komatsu Y et al Nucleic Acids Res 25 3889 3894 1997 Kobayashi N et al J Invest Dermatol 101 685 689 1993 0 Mori T et al Photochem Photobiol 54 225 232 1991 OoN NMNHBWN Related Products CycLex Cellular Histone Acetylation Assay Kit Cat CY 1140 CycLex Cellular UV DNA Damage Detection Kit Cat CY 1141 CycLex BrdU Cellul
3. use in diagnostic procedures ernational Corporation Note 1 Complete removal of liquid at each step is essential to good performance After the lastwash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels Note 2 Although we suggest to conduct experiments as outlined above the optimal experimental conditions will vary depending on the parameters being investigated and must besdetermined by the individual user Especially an appropriate cell number must be optimized for your experiment Troubleshooting The signals and backgrounds are influenced a great deal by cell line and cell number that you plated please ensure the appropriate cell number for your experiment Please conduct the experiment to optimize cell number for this assay See Example of Test Results Fig 2 and Fig 4 2 Unavoidable background no UV irradiation is observed even ifan appropriate cells number is used It is usually around 0 3 0 4 See Example of Test Results Fig 1 and 3 3 With some cell lines higher cell concentrations more ham1 x 10 cells well in case of adherent cells may lead to increasing absorbance values in no UV irradiation background rather than those in UV irradiation 4 All UV irradiation including no UV irradiation control should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures signi
4. 420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com yy y C l 9 10 11 12 13 14 15 16 IA Cellular UV DNA Damage Detection Kit cLex User s Manual A Bi For Research Use Only Not for use in diagnostic procedures ernational Corporation Detection of Signals Addition of Primary and Secondary Antibodies and Substrate Reagent Add 200 uL well of Blocking Solution and incubate for at least 30 minutes at room temperature ca 25 C Remove Blocking Solution with a wrist flick Add 50 uL well of Primary Antibody Solution and incubate for 1 hour at room temperature ca 25 C Alternatively this incubation period can be varied between 30 120 minutes depending on individual requirements Remove Primary Antibody Solution with a wrist flick Rinse the wells once with 200ul well of Wash Buffer Remove Wash Buffer with a wrist flick While still inverted tap the plate onto absorbent paper Wash wells 4 times with 200 wL well of Wash Buffer for 2aninutes each with shaking at ca 200 rpm on an orbital microplate shaker Remove Wash Bufferan between each wash with a wrist flick Add 50 uL well of Secondary Antibody Solution and incubate for 1 hour at room temperature ca 25 C Remove Secondary Antibody Solution with a wristflick Rinse wells once with 200 uL well of Wash Buffer Remove Wash Buffer with wrist flick and t
5. 8 0 8 N N 0 6 z 0 6 0 4 0 4 0 2 0 2 0 0 0 0 1 10 100 0 1 10 100 J m2 CPDs 50 000 cells well HeLa 1 2 1 0 0 8 TA 0 6 0 4 0 2 0 0 0 1 10 100 J m2 Cat CyY 1141 11 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit User s Manual KI Bi For Research Use Only Not for use in diagnostic procedures mernational Corporation wy YCLEX Fig 2 Effect of cell number on ELISA value using HeLa cells CPDs HeLa O UV0 UV 0 156J m2 UV 0 625J m2 m UV 2 5J m2 A UV 10J m2 A amp UV 40J m2 12 500 25 000 Cell number Cat CY 1141 12 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit User s Manual KI Bi x For Research Use Only Not for use in diagnostic procedures mernational Corporation Fig 3 UV dose dependency using Raji cells CPDs 12 500 cells well Raji CPDs 25 000 cells well Raji 1 4 1 2 1 0 gt 0 8 N 0 6 0 4 0 2 0 0 0 1 Im 10 CPDs 50 000 cells well Raji 1 4 1 2 1 0 0 8 N 0 6 0 4 0 2 0 0 0 1 10 100 J m2 Cat CyY 1141 13 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit User s Manual KI Bi
6. CPDs on DNA CPDs in the cell is detected by anti CPDs antibody During UV irradiation is carried out on wells of the microtiter plate CPDs will be formed on the DNA of living cells To enable antibody binding to the CPDs on the genomic DNA cells must be fixed permeabilized and the DNA denatured Detector anti CPDs antibody is pipetted into the wells and allowed to incubate for on hour during which time it binds to any CPDs Unbound antibody is washed away and horseradish peroxidase conjugated secondary antibody is added which binds to the detector antibody The horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TMB from a colorless solution to a blue solution or yellow after the addition of stopping reagent The color is quantified by spectrophotometry and reflects the relative amount of CPDs in the cells Cat CY 1141 2 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit User s Manual Bi For Research Use Only Not for use in diagnostic procedures ernational Corporation wy YCLEX The CycLex Cellular UV DNA Damage Detection Kit is designed to measure the relative levels of cyclobutane pyrimidine dimers on genomic DNA in situ The summary of the assay is shown in below I Summary of Procedure for adherent cells Culture adherent cells in microplate at 1 4 x 10 cells we
7. DNA purified from cultured cells or from the skin epidermis using an enzyme linked immunosorbent assay ELISA and to visualize and measure phOtoproducts in DNA in cultured cells or the skin using indirect immunofluorescence IIF This technology would contribute to understanding of molecular mechanisms of cellular responses to UV and DNA damage in the photobiology and the pigment cell biology A rapid and convenient method for evaluating the formation of cyclobutane pyrimidine dimers CPDs in a cell after UV irradiation was developed which detects CPDs adducts on the genomic DNA by means of anti CPDs antibody in a cellular ELISA format It has been shown that a precise evaluation of CPDs formation could be performed by the measurement of CPDs adducts in purified genomic DNA by ELISA In addition there is a good reverse proportion betweensthe cellular CPDs ELISA value and ability of nucleotide excision repair NER whens the cell are incubate for several hours after UV irradiation for repair as shown for a variety of NER deficient cell lines such as xeroderma patient derived cell lines The CycLex Cellular UV DNA Damage Detection Kit can be used in many different in vitro cell systems without any purification step of genomic DNA Principle of the Assay The CycLex Cellular UV DNA Damage Detection Kit based on the formation of CPDs on the genomic DNA after UV irradiation in thescells that are cultured in microtiter plates After formation of
8. Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit oy GA cLex User s Manual y For Research Use Only Not for use in diagnostic procedures mernational Corporation Detailed Protocol The CycLex Cellular UV DNA Damage Detection Kit includes all reagents except cell culture microplate for detection of CPDs formed in genomic DNA in cultured cells Since experimental conditions may vary treatment cells with test compound should be assayed in duplicate Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Reagents All reagents need to be brought to room temperature prior to the assay Assay reagents are supplied ready to use with the exception of 10X Wash Buffer 20X Primary Antibodyi Solution and 20X Secondary Antibody Solution 1 Prepare a working solution of Wash Buffer by adding 100 mL of the LOX Wash Buffer provided to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Prepare a working solution of Primary Antibody Solution Dilute 20X Primary Antibody Solution 1 20 with Primary Antibody Dilution Buffer Primary Antibody Solution can be stored for up to one week protected from light at 4 C Prepare appropriate volume for your assay You will need 50 60 uL of Primary Antibody Solution per assay well Disc
9. aining 600 uk of HRP horseradish peroxidase conjugated Secondary Antibody Secondary Antibody Dilution Buffer One bottle containing 12 mL of Secondary Antibody Dilution Buffer Ready to use Substrate Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 NHySO Ready to use Sulfuric Acid is a strong acid Wear disposable gloves and eve protection whenghandling Materials Required but not Provided e 96 well microplate tissue culture grade flat bottom e UV crosslinker or irradiator Stratalinker 1800 Stratagene etc e Centrifuge with rotor for icroplate for suspension cell only e Cell culture media e 1X PBS pH 7 2 e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips e Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer e Microplate washer optional Manual washing is possible but not preferable e Software package facilitating data generation and analysis optional e Reagent reservoirs e Deionized water of the highest quality e Absorbent paper disposable paper towels Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength of 450 nm which will give a
10. ap plate onto absorbent paper Wash wells 4 times with 200 wk wellhof Wash Buffer for 2 minutes each with shaking at ca 200 rpm on an orbital microplate shaker Remove Wash Buffer in between each wash with a wrist flick After last wash with Wash Buffer rinse wells once with 300 wl well of 1X PBS Remove with a wrist flick and tap onto absorbent paper Ensure that that no liquid remains in the well Add 50 uL well of Substrate Reagent Avoid exposing the microplate to direct sunlight covering the plate with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed Incubate the plate for 15 25 minutes at room temperature ca 25 C The incubation time may be extended up to 30 minutes if the reaction temperature is below than 20 C Add 50 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Cat CY 1141 9 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit oy aA cLex User s Manual A Bi y For Research Use Only Not for
11. ar ELISA Kit Cat CY 1142 CycLex Histone H2A X Phosphorylation Cellular ELISA Kit Cat CY 1143 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cy lex co jp CycLex CircuLex prod cts are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercialise please contact us via email Cat CY 1141 15 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com
12. ard any unused Primary Antibody Solution after diluted 3 Prepare a working solution of Secondary Antibody Solution Dilute 20X Secondary Antibody Solution 1 20 with Secondary Antibody Dilution Buffer Secondary Antibody Solution can be stored for up to one week protected from dight at 4 C Prepare appropriate volume for your assays You will need 50 60 uL of Secondary Antibody Solution per assay well Discard any unused Secondary Antibody Solution after diluted Cat CY 1141 7 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com yy y Cellular UV DNA Damage Detection Kit cLex User s Manual A Bi For Research Use Only Not for use in diagnostic procedures ernational Corporation Assay Procedure A Culture cells in 96 well microplate and UV irradiation l Plate adherent cells into a 96 well microplate at 1 4 x 10 cells well in a final volume of 100 uL well For most experiments 2 5 x 10 cells well are sufficient and adequate for most adherent cells 1 2 x 10 cells well are sufficient and adequate for most suspension cells See Fig 1 4 Incubate the microplate at 37 C over night in CO incubator Remove the culture media Add 50 uL of sterile PBS in well Perform UV irradiation to each well No irradiation control should be run in duplicate as a negative control 0 0 1 0 2 0 5 1 5 10 40 im Remove the PBS A
13. cyclobutane pyrimidine dimers in cells 4 Study on the repair mechanisms of UV induced DNA damage by the nucleotide excision repair in cells This assay kit_is for research use only and not for use in diagnostic or therapeutic procedures Storage Upon receipt store all components at 4 C e Don t expose reagents to excessive light Cat CY 1141 1 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Pry Cellular UV DNA Damage Detection Kit od cLex User s Manual pa Bi y For Research Use Only Not for use in diagnostic procedures mernational Corporation Introduction DNA damage in cells exposed to ultraviolet UV radiation plays significant roles in cell cycle arrest activation of DNA repair cell killing mutation and neoplastic transformation The major types of DNA damage induced by UVB 280 315 nm component of sunlight and by UVC 200 280 nm are cyclobutane pyrimidine dimers CPDs and 6 4 photoproducts 6 4PPs which are formed between adjacent pyrimidine nucleotides on the same strand of DNA Approximately 70 80 of UV induced DNA damage is CPDs and the remaining is 6 4PPs and Dewar isomer of 6 4PPs These types of DNA lesions are repaired by nucleotide excision repair NER system in normal human cells Moriet al 10 have established monoclonal antibodies specific for CPDs or 6 4PPs These antibodiesyenable one to quantitate photoproducts in
14. dd 100 uL of the culture medium in well Incubate the microplate at 37 C for appropriate time im CO incubator in case of repair experiment Note Although we suggest to conduct experiments as outlined above the optimal experimental conditions will vary depending on the_ parameters being investigated and must be determined by the individual user Especially an appropriate cell number must be optimized for your experiment B Fixing and denaturing the cells to 96 well plate Fixing and denaturing the cells in the 96 well plates should be done as soon as the desired incubation has completed For adherent cells 1 Remove media from wells with a wrist flick Avoid touching the bottom of the well and removing cells For suspension cells 1 Remove media from wells by centrifugation the 96 well microplate at 2 100 x g for 10 min and then suction using a canulla Avoid touching the bottom of the well and removing cells Dry the cells using a hair dryenfor about 15 minutes After this step the dried cells can be stored for up to one week at 4 C 2 Add 200 wL well of Fixing Denaturing Solution slowly to insure cells are not detached from the plastic Let stand for 30 minutes at room temperature ca 25 C 3 Remove Fixing Denaturing Solution from wells with a wrist flick While still inverted tap the plate gently onto absorbent paper to remove any excess fixing agent still within the wells Cat CY 1141 8 Version 120
15. f Blocking Solution y Incubate 30 min at room temp Discard the Blocking Solution v Add 50 uloof Primary Antibody Anti CPDs antibody Incubate hr at room temp Wash the wells Add 50 uL of HRP conjugated Secondary Antibody Y Incubate 1 hr at room temp Wash the wells Add 50 uL of Substrate Reagent Add 50 uL of Stop Solution v Measure absorbance at 450 nm Cat CY 1141 4 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit wy GA cLex User s Manual Pa Bi y For Research Use Only Not for use in diagnostic procedures mernational Corporation Materials Provided All compounds treatment should be assayed in duplicate The following components are supplied and are sufficient for 200 assays Fixing Denaturing Solution One bottle containing 50 mL of Fixing Denaturing Solution Ready to use The solution is a strong alkaline Wear disposable gloves and eye protection when handling Blocking Solution One bottle containing 50 mL of Blocking Solution Ready to use 10X Wash Buffer One bottle containing 100 mL of 10X Wash Buffer containing 2 fween 20 20X Primary Antibody Solution One vial containing 600 uL of anti cyclobutane pyrimidine dimers antibody Primary Antibody Dilution Buffer One bottle containing 12 mL of Primary Antibody Dilution Buffer Ready to use 20X Secondary Antibody Solution One vial cont
16. ficantly different from those specified may give erroneous results 5 Poor duplicates accompanied by elevated values for wells containing no UV irradiation control indicate insufficient washing or vigorous washing Wash the plate thoroughly and gently 6 Overall low signal may indicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent Stability All of the reagents included in the CycLex Cellular UV DNA Damage Detection Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C For long term storage of 20X Primary Antibody Solution and 20X Secondary Antibody Solution it is recommended to store at below 70 C For research use only not for use in diagnostic or therapeutic procedures Cat CY 1141 10 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com kai ycLex a Cellular UV DNA Damage Detection Kit User s Manual For Research Use Only Not for use in diagnostic procedures MBL International Corporation Example of Test Results Fig l UV dose dependency using HeLa cells CPDs 12 500 cells well HeLa CPDs 25 000 cells well HeLa 1 2 1 2 1 0 1 0 0
17. ll Y Incubate O N at 37 C in CO incubator UV irradiate the cells y Incubate an appropriate time at 37 C in CO incubator Discard the culture medium v Add 200 uL of Fixing Denaturing solution Y Stand for 30 min at room temp Discard the Fixing Denaturing solution v Add 200 uL of Blocking Solution Y Incubate 30 min at room temp Discard the Blocking Solution v Add 50 uL of Primary Antibody Anti CPDs antibody Incubate 1 hr at room temp Wash the wells Add 50 uloof HRP conjugated Secondary Antibody Yy Incubate 1 hr at room temp Wash the wells Add 50 uL of Substrate Reagent Add 50 uL of Stop Solution v Measure absorbance at 450 nm Cat CY 1141 3 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit User s Manual Bi For Research Use Only Not for use in diagnostic procedures mernational Corporation wy YCLEX II Summary of Procedure for suspension cells Culture suspension cells in microplate at 1 2 x 10 cells well Y Incubate O N at 37 C in CO incubator UV irradiate the cells y Incubate an appropriate time at 37 C in CO24neubat r c f g the microplate at 3 500 rpm for 10 min v Discard the culture medium Y Air dry the cells by hair dryer for 15 min v Add 200 uL of Fixing Denaturing Solution Y Stand for 30 min at room temp Discard the Fixing Denaturing Solution v Add 200 uL o
18. somewhat higher reading Cat CY 1141 5 Version 120420 15A Constitution Way Woburn MA 01801 Phone 1 800 200 5459 Fax 781 939 6963 mblintl com Cellular UV DNA Damage Detection Kit oy GA cLex User s Manual y For Research Use Only Not for use in diagnostic procedures mernational Corporation Precautions and Recommendations Safety Warnings and Precautions The CycLex Cellular UV DNA Damage Detection Kit is designed for research use only and not recommended for internal use in humans or animals All chemicals should be considered potentially hazardous and principles of good laboratory practice should be followed Technical Notes 1 When performing washes manually avoid introducing bubbles when dispensing liquids intosthe wells and ensure each well is filled with buffer but not overflowing to avoid cross contamination between wells Empty wells with a wrist flick motion over an appropriate receptacle and while still inverted blot any remaining moisture onto clean absorbent paper 2 Agitation of wells during incubation of Blocking Buffer and Antibody steps 18 recommended to reduce non specific background If microtiter plate agitator is not available a platformpyortex at a low setting can be used e g level 1 of Fisher s Genie II platform vortex If background problems occur simply increase the number and or duration of washes 3 A brief 1X PBS rinse is recommended prior to the addition of the HRP substra
19. te to remove any traces of the Tween 20 with can interfere with the HRP activity 4 Do not allow the wells to dry out during the protocol 5 Incubation temperatures for Primary Antibody and Detectiom Antibody can be varied and should be empirically determined General Notes e Allow all the components to come to room temperature before use e Do not use kit components beyond the indicated kit expiration date e Rinse all detergent residue from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits e The buffers and reagents used in this kit contain NaN as preservatives Care should be taken to avoid direct contact with these reagents e Dispose of tetra methylbenzidine TMB containing solutions in compliance with local regulations e Avoid contact with thesalkaline Fixing Denaturing Solution the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves andieye protection when handling immunodiagnostic materials and samples of human origin and these reagents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary e CAUTION Sulfuric Acid is a strong acid Fixing Denaturing Solution is a strong alkaline Wear disposable gloves and eye protection when handling Fixing Denaturing Solution and Stop Solution Cat CY 1141 6 Version 120420 15A Constitution Way
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