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HTS01 BrdU Cell Proliferation Assay, HTS

BrdU Cell Proliferation Assay, HTS MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Bromodeoxyuridine Assay

HTS01
  
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Fluorometric
      Description
      Overview

      This product has been discontinued.





      High-throughput screening version of Cat. No. QIA58. A non-isotopic immunoassay for the quantitation of BrdU incorporation into newly synthesized DNA of actively proliferating cells. Wavelengths between 315 and 340 nm for excitation and 370 and 470 for emission can be used for detection.
      Note: 1 T = 1 test.
      Catalogue NumberHTS01
      Brand Family Calbiochem®
      SynonymsBromodeoxyuridine Assay
      Materials Required but Not Delivered• 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • 500 ml graduated cylinder
      • PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4-7H2O, 1.4 mM KH2PO4)
      • Deionized or distilled H2O
      • Fluorometer (Fluorescent Plate Reader) capable of measuring excitation ~325 nm and emission ~420 nm. Wavelengths between 315 and 340 nm for excitation and 370 and 470 for emission can be used for detection.
      • Tissue culture plate (96 well culture dish) suitable for use in a fluorometer, such as Corning Costar Cat No. 3603 (black, clear bottom)
      • Reagent trough
      • Syringe filter (0.2 mm)
      • Syringe
      References
      ReferencesHardonk, M.J. and G. Harms, 1990. Acta histochemica, Suppl. 39, 99.
      Muir, D., et al. 1990. Analytical Biochemistry 185, 377.
      Lanier, T. L., et al. 1989. Carcinogenesis 10, 1341.
      Magaud, J.-P., et al. 1988. J. Immunol. Methods 106, 95.
      Collins, S.J. 1987. Blood 70, 1233.
      Porstmann, et al. 1985. J. Immunol. Methods 82, 169.
      Raza, A., et al. 1985. Cytometry 6, 633.
      Morstyn, G., et al. 1983. J. Clin. Invest. 72, 1844.
      Gratzner, H.G., 1982. Science 218, 474.
      Product Information
      Detection methodFluorometric
      Form200 or 1000 Tests
      Format96-well plate
      Kit containsBrdU Label, Fixative/Denaturing Solution, Anti-BrdU Antibody, Antibody Diluent, Peroxidase Goat Anti-Mouse IgG, Conjugate Diluent, Fluorogenic Substrate, 20X Plate Wash Concentrate, Stop Solution, and a user protocol.
      Applications
      Biological Information
      Assay time3 h
      Sample TypeIntact Cells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 11-20/21/22-36/38-46-61

      Highly flammable.
      Harmful by inhalation, in contact with skin and if swallowed.
      Irritating to eyes and skin.
      May cause heritable genetic damage.
      May cause harm to the unborn child.
      S PhraseS: 26-36/37/39-45-7-16

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Keep container tightly closed.
      Keep away from sources of ignition - No Smoking.
      Product Usage Statements
      Intended useThe BrdU Proliferation Assay (Cat. No. HTS01) is a non-isotopic immunoassay for the quantitation of bromodeoxyuridine incorporation into newly synthesized DNA of actively proliferating cells.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage -20°C
      Storage ConditionsUpon arrival store the entire contents of the kit at -20°C in a non-frostfree freezer. BrdU Proliferation Assay kit components are shipped on dry ice.
      Protect from Moisture Protect from moisture
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsBrdU Label, Fixative/Denaturing Solution, Anti-BrdU Antibody, Antibody Diluent, Peroxidase Goat Anti-Mouse IgG, Conjugate Diluent, Fluorogenic Substrate, 20X Plate Wash Concentrate, Stop Solution, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalog Number GTIN
      HTS01 0

      Documentation

      BrdU Cell Proliferation Assay, HTS Certificates of Analysis

      TitleLot Number
      HTS01

      References

      Reference overview
      Hardonk, M.J. and G. Harms, 1990. Acta histochemica, Suppl. 39, 99.
      Muir, D., et al. 1990. Analytical Biochemistry 185, 377.
      Lanier, T. L., et al. 1989. Carcinogenesis 10, 1341.
      Magaud, J.-P., et al. 1988. J. Immunol. Methods 106, 95.
      Collins, S.J. 1987. Blood 70, 1233.
      Porstmann, et al. 1985. J. Immunol. Methods 82, 169.
      Raza, A., et al. 1985. Cytometry 6, 633.
      Morstyn, G., et al. 1983. J. Clin. Invest. 72, 1844.
      Gratzner, H.G., 1982. Science 218, 474.
      User Protocol

      Revision17-June-2011 RFH
      SynonymsBromodeoxyuridine Assay
      Form200 or 1000 Tests
      Format96-well plate
      Detection methodFluorometric
      StorageUpon arrival store the entire contents of the kit at -20°C in a non-frostfree freezer. BrdU Proliferation Assay kit components are shipped on dry ice.
      Intended useThe BrdU Proliferation Assay (Cat. No. HTS01) is a non-isotopic immunoassay for the quantitation of bromodeoxyuridine incorporation into newly synthesized DNA of actively proliferating cells.
      BackgroundEvaluation of cell cycle progression is essential for investigations in many scientific fields. Measurement of [3H] thymidine incorporation as cells enter S phase has long been the traditional method for the detection of cell proliferation. Subsequent quantification of [3H] thymidine is performed by scintillation counting or autoradiography. This technology is slow, labor intensive and has several limitations including the handling and disposal of radioisotopes and the necessity of expensive equipment. A well-established alternative to [3H] thymidine uptake has been demonstrated by numerous investigators. In these methods bromodeoxyuridine (BrdU), a thymidine analog, replaces [3H] thymidine. BrdU is incorporated into newly synthesized DNA strands of actively proliferating cells. Following partial denaturation of double stranded DNA, BrdU is detected immunochemically allowing the assessment of the population of cells, which are actively synthesizing DNA. This BrdU Proliferation Assay (Cat. No. HTS01) involves incorporation of BrdU into cells cultured in microtiter plates and BrdU immunolabeling using the cell layer as the solid phase. The resultant assay is sensitive, rapid, easy to perform and applicable to high sample throughput. In addition to evaluation of cell proliferation, information such as cell number, morphology and analysis of cellular antigens can be obtained from a single culture.
      Principles of the assayThe BrdU Proliferation Assay (Cat. No. HTS01) involves incorporation of BrdU into cells cultured in microtiter plates and BrdU immunolabeling using the cell layer as the solid phase. During the final 2 to 24 h of culture BrdU is added to wells of the microtiter plate. BrdU will be incorporated into the DNA of dividing cells. To enable antibody binding to the incorporated BrdU, cells must be fixed, permeabilized and the DNA denatured. This is all done in one step by treatment with Fixative/Denaturing Solution. Detector anti-BrdU monoclonal antibody is pipetted into the wells and allowed to incubate for one hour, during which time it binds to any incorporated BrdU. Unbound antibody is washed away and horseradish peroxidase-conjugated goat anti-mouse is added, which binds to the detector antibody.
      The horseradish peroxidase catalyzes the conversion of the fluorogenic substrate to a blue fluorescent product the intensity of which is proportional to the amount of incorporated BrdU in the cells. The blue fluorescent product is quantified using a fluorometer.
      Materials providedThe BrdU Proliferation Assay is provided in either a 200 test kit or a 1000 test kit.

      • BrdU LABEL (Kit Component No. JA1595-50UL): A 2000X solution of BrdU.
      • BrdU FIXATIVE/DENATURING SOLUTION (Kit Component No. JA1598-25ML):
      • 100X ANTI-BrdU ANTIBODY (Kit Component No. JA1599-1ML): Stock solution of the antibody.
      • ANTIBODY DILUENT (Kit Component No. JA)1604-100ML: Solution for dilution of the anti-BrdU antibody.
      • BrdU PEROXIDASE GOAT ANTI-MOUSE IgG (Kit Component No. JA1618-50UG): Refer to vial label for lot specific dilution.
      • BrdU CONJUGATE DILUENT (Kit Component No. JA1615-100ML): Buffer for dilution of Peroxidase Goat Anti-Mouse IgG.
      • BrdU FLUOROGENIC SUBSTRATE AND BrdU FLUOROGENIC PEROXIDE SOLUTION (Kit Component No. JA1910-120ML): A Fluorogenic Substrate Working Stock Solution is prepared by mixing 9 parts Substrate to 1 part Peroxide Solution.
      • Fluorogenic Peroxide Solution (Kit Component No. JA1911-12ML):
      • BrdU 20X PLATE WASH CONCENTRATE (Kit Component No. JA1617-100ML): 20X concentrated solution of PBS and surfactant. Contains 2% chloroacetamide.
      • BrdU FLUOROGENIC STOP SOLUTION (Kit Component No. JA1912-120ML):
      Materials Required but not provided• 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • 500 ml graduated cylinder
      • PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4-7H2O, 1.4 mM KH2PO4)
      • Deionized or distilled H2O
      • Fluorometer (Fluorescent Plate Reader) capable of measuring excitation ~325 nm and emission ~420 nm. Wavelengths between 315 and 340 nm for excitation and 370 and 470 for emission can be used for detection.
      • Tissue culture plate (96 well culture dish) suitable for use in a fluorometer, such as Corning Costar Cat No. 3603 (black, clear bottom)
      • Reagent trough
      • Syringe filter (0.2 mm)
      • Syringe
      Precautions and recommendations• Do not expose reagents to excessive light.
      • Wear disposable gloves and eye protection.
      • Do not mix reagents from different kits.
      • Do not mouth pipette or ingest any of the reagents.
      • The buffers and reagents used in this kit contain anti-microbial and anti-fungal reagents. Care should be taken to prevent direct contact with these products.
      • Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.
      • Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
      Reagent preparationBefore first use: 1. Remove the Fixative/Denaturing Solution and place at room temperature for at least 4 h prior to use. Precipitates that may occur while cold should go back into solution. 2. Reconstitute the Peroxidase Goat Anti-Mouse IgG with the appropriate volume of PBS. • For the 200 test kit, reconstitute Peroxidase Goat Anti-Mouse IgG in 125 µl of PBS • For the 1000 test kit, reconstitute Peroxidase Goat Anti-Mouse IgG in 250 µl of PBS Allow solution to stand at room temperature for 10 min. (or until solution is clear). Divide into small aliquots; aliquots not to be used that same day should be stored at -20°C in a non-frostfree freezer. Once the kit components have been thawed for use: • The BrdU label and 100X Anti-BrdU Antibody should be divided into small aliquots and stored at -20°C in a non-frostfree freezer with the Peroxidase Goat Anti-Mouse IgG; avoid multiple freeze/thaw cycles. • All the other kit components (Substrate, Peroxide Solution, Antibody Diluent, Conjugate Diluent, 20X Plate Wash Concentrate, Fixative/Denaturing Solution, Stop Solution) should be stored at 4°C.
      Detailed protocol1. The BrdU Proliferation Assay is provided with all reagents necessary to run 200 (or 1000) tests or microtiter plate wells. Replicate samples should be assayed due to variation in biological responses at the cellular level. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.
      2. Seed 100 µl of cells at 104 to 106 cells/ml into a 96 well culture dish. If adherent cells are being used, allow cells to attach prior to treatment with test substance (i.e. proliferation inducer or inhibitor). Two types of controls should also be set up to insure validity of the experiment.
      I. Blank: Add only tissue culture media (no cells).
      II. Background: Cells are present in the wells but do not add the BrdU Label. Follow protocol as usual from step 6.
      3. Prepare a working stock of BrdU by diluting the BrdU Label 1:2000 into fresh tissue culture media (for example, add 2 µl of BrdU Label to 4 ml fresh tissue culture media).
      4. Pipette 20 µl of this working stock to each well to be labeled.
      5. Allow BrdU to incubate with cells for 2-24 h in the tissue culture incubator (Figure 1). If non-adherent cells are being used, centrifuge 96 well dish for 10 min at 1000 rpm.
      6. Remove contents of wells by inverting over sink and blot gently on paper towels.
      7. Add 200 µl of the Fixative/Denaturing Solution to each well.
      8. Incubate for 30 min at room temperature.
      9. Remove contents of wells by inverting over sink and tapping on paper towels. Plates may be stored after this step at 4°C for 1 week.
      10. Dilute the 100X Anti-BrdU Antibody 1:100 in the Antibody Dilution Buffer (i.e. 120 µl to 12 ml of Antibody Diluent). Pipette 100 µl of this solution to each well and incubate for 1 h at room temperature.
      11. Prepare a working solution (1X) of Wash Buffer by adding 25 ml of the 20X concentrated solution (provided), to 475 ml of deionized water. Mix well.
      12. Wash wells 3 times with automatic plate washer or by hand with 1X Wash Buffer making sure each well is filled completely. Blot the plate gently on paper towels.
      13. Prepare the conjugate by diluting the reconstituted Peroxidase Goat Anti-Mouse IgG HRP Conjugate in the Conjugate Diluent and syringe filter through 0.2 micron filter. NOTE: Dilution of conjugate is lot specific. Refer to vial label for lot specific dilution.
      14. Pipette 100 µl of this solution into each well and incubate for 30 min at room temperature.
      15. Prepare a Fluorogenic Substrate Working Stock Solution by mixing 9 parts Fluorogenic Substrate to 1 part Fluorogenic Peroxide Solution. The Working Stock Solution is stable for 24 h at room temperature and no protection from light is required. Prepare only the amount of Working Stock Solution that will be used within 24 h.
      16. At the end of the 30 min incubation (step 14), wash wells 3 times with automatic plate washer or by hand with 1X Wash Buffer making sure each well is filled completely.
      17. FLOOD ENTIRE PLATE WITH dH2O. Remove contents of wells by inverting over sink and tapping on paper towels.
      18. Add 100 µl of Fluorogenic Substrate Working Stock Solution (prepared in step 15.) to each well and incubate at room temperature for 30 min.
      19. Add 100 µl of Stop Solution to each well in the same order as the previously added Substrate Solution.
      20. Measure plate on a Fluorometer capable of measuring excitation ~325 nm and emission ~420 nm. (The assay was validated with a BMG FLUOstar Fluorometer with a 320 nm excitation and a 460 nm emission filter). Wavelengths between 315 and 340 nm for excitation and 370 and 470 nm for emission can be used for detection. Wells must be read within 30 min of adding the Stop Solution.
      Assay characteristics and examplesThe data can be expressed as the relative signal (RFU) after subtraction of the relative signal of the appropriate buffer controls (Figure 2 and 3). The data may also be expressed as a ratio of signal obtained from a labeled sample (+BrdU) to an unlabeled sample (-BrdU) after subtraction of the appropriate endogeneous fluorescence or buffer controls (Figure 3).

      Figure 1: Two vs. 24 h BrdU labeling of two cell lines at two cell concentrations


      Figure 2: Model system

      Cellular Proliferation of Human Peripheral Blood Lymphocytes After Stimulation With Different Doses of PHA Detected By the BrdU Proliferation Assay (Cat. No. HTS01). Proliferation Assay (Cat No. HTS01) involves incorporation of bromodeoxyuridine (BrdU) into cells cultured in microtiter plates and BrdU immunolabeling using the cell layer as the solid phase. During the final 2 to 24 h of culture BrdU is added to wells of the microtiter plate. BrdU will be incorporated into the DNA of dividing cells. To enable antibody binding to the incorporated BrdU, cells must be fixed, permeabilized and the DNA denatured. This is all done in one step by treatment with Fixative/Denaturing Solution. Detector anti-BrdU monoclonal antibody is pipetted into the wells and allowed to incubate for one hour, during which time it binds to any incorporated BrdU. Unbound antibody is washed away and horseradish peroxidase-conjugated goat anti-mouse is added, which binds to the detector antibody. In the presence of hydrogen peroxide, the HRP catalyzes the conversion of the substrate to produce a highly fluorescent product the signal of which is proportional to the amount of incorporated BrdU in the cells.


      Figure 3: Signal/Noise Ratio and Signal at 320 Excitation/405 Emission vs. 320 Excitation/

      At 405 nm emission, the signal is increased. At 460 nm emission, the ratio of BrdU labeled to the no BrdU control is generally higher. The cells were label with BrdU for 24 h, the no cell controls were not subtracted from the data.


      Figure 4: Sensitivity

      With a 24 h BrdU labeling 600 plated cells (~1,800 growing cells per well) can easily be distinguished from background.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.