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QIA58 BrdU Cell Proliferation Assay

This proliferation assay is a non-isotopic immunoassay for quantification of BrdU incorporation into newly synthesized DNA of actively proliferating cells.

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

Synonyms: Bromodeoxyuridine Assay

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QIA58
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Overview

Replacement Information

Key Spec Table

Detection Methods
Colorimetric

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      QIA58-200TEST
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          Description
          OverviewBrdU Cell Proliferation Assay is a non-isotopic immunoassay for quantification of BrdU incorporation into newly synthesized DNA of actively proliferating cells. It is sensitive, rapid, and easy to perform.
          Note: 1 T = 1 test.
          Catalogue NumberQIA58
          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
          • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450-540 or 450-595 nm
          • Tissue culture plate (96 well culture dish)
          • Reagent trough
          • Micro filter (0.2 µm)
          • Syringe
          References
          ReferencesHardonk, M.J. and Harms, G. 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, T., 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 methodColorimetric
          Form200 or 1000 Tests
          Format96-well plate
          Kit containsBrdU Label, Fixative/Denaturing Solution, BrdU Antibody, Antibody Diluent, Peroxidase Goat Anti-Mouse IgG, Conjugate Diluent, Substrate, Plate Wash Concentrate, Stop Solution, and a user protocol.
          Quality LevelMQ100
          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: 11-20/21/22-35-36/38-46-61

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

          Keep container tightly closed.
          Keep away from sources of ignition - No Smoking.
          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).
          Product Usage Statements
          Intended useThe Calbiochem® BrdU Cell Proliferation Assay 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.

          Prior to 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 1X PBS.
          • For the 200 test kit, reconstitute Peroxidase Goat Anti-Mouse IgG in 125 µl of 1X PBS. Use Conjugate Diluent for any further lot-specific dilution.
          • For the 1000 test kit, reconstitute Peroxidase Goat Anti-Mouse IgG in 250 µl of 1X PBS. Use Conjugate Diluent for any further lot-specific dilution.
          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, Antibody Diluent, Conjugate Diluent, 20X Plate Wash Concentrate, Fixative/Denaturing Solution, Stop Solution) should be stored at 4°C.
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsBrdU Label, Fixative/Denaturing Solution, BrdU Antibody, Antibody Diluent, Peroxidase Goat Anti-Mouse IgG, Conjugate Diluent, Substrate, Plate Wash Concentrate, Stop Solution, and a user protocol.
          Specifications
          Global Trade Item Number
          Catalogue Number GTIN
          QIA58-1000TEST 04055977209129
          QIA58-200TEST 04055977209136

          Documentation

          BrdU Cell Proliferation Assay MSDS

          Title

          Safety Data Sheet (SDS) 

          BrdU Cell Proliferation Assay Certificates of Analysis

          TitleLot Number
          QIA58

          References

          Reference overview
          Hardonk, M.J. and Harms, G. 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, T., 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

          Revision06-June-2016 MJ
          SynonymsBromodeoxyuridine Assay
          Form200 or 1000 Tests
          Format96-well plate
          Detection methodColorimetric
          StorageUpon arrival store the entire contents of the kit at -20°C, in a non-frostfree freezer.

          Prior to 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 1X PBS.
          • For the 200 test kit, reconstitute Peroxidase Goat Anti-Mouse IgG in 125 µl of 1X PBS. Use Conjugate Diluent for any further lot-specific dilution.
          • For the 1000 test kit, reconstitute Peroxidase Goat Anti-Mouse IgG in 250 µl of 1X PBS. Use Conjugate Diluent for any further lot-specific dilution.
          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, Antibody Diluent, Conjugate Diluent, 20X Plate Wash Concentrate, Fixative/Denaturing Solution, Stop Solution) should be stored at 4°C.
          Intended useThe Calbiochem® BrdU Cell Proliferation Assay is a non-isotopic immunoassay for the quantitation of bromodeoxyuridine incorporation into newly synthesized DNA of actively proliferating cells.
          BackgroundA non-isotopic enzyme immunoassay for the quantification of cell proliferation. Evaluation 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. The Calbiochem® BrdU Cell Proliferation Assay involves incorporation of BrdU into cells cultured in 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 Calbiochem® BrdU Cell Proliferation Assay involves incorporation of BrdU into cells cultured in 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 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 1 h, 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 chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of incorporated BrdU in the cells. The colored reaction product is quantified using a spectrophotometer.
          Materials providedThe BrdU Cell Proliferation Assay is provided in either a 200 test kit or a 1000 test kit.

          • BrdU LABEL (Kit Component No. JA1595): A 2,000-fold solution of BrdU.
          • Fixative/Denaturing Solution (Kit Component No. JA1598)
          • 100X Anti-BrdU Antibody (Kit Component No. JA1599): Stock solution of the antibody.
          • Antibody Diluent (Kit Component No. JA1604): Solution for dilution of the anti-BrdU antibody.
          • Peroxidase Goat Anti-Mouse IgG (Kit Component No. JA1618): Lyophilized. Refer to page 1 for reconstitution.
          • Conjugate Diluent (Kit Component No. JA1615): For dilution of reconstituted Peroxidase Goat anti-mouse IgG peroxidase conjugate.
          • Substrate (Kit Component No. JA1608): Tetra-methylbenzidine solution.
          • 20X Plate Wash Concentrate (Kit Component No. JA1617): 20X concentrated solution of PBS and surfactant. Contains 2% chloroacetamide.
          • Stop Solution (Kit Component No. JA1616): 2.5N sulfuric acid.
          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
          • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450-540 or 450-595 nm
          • Tissue culture plate (96 well culture dish)
          • Reagent trough
          • Micro filter (0.2 µm)
          • 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.
          Detailed protocol1. The BrdU Cell Proliferation Assay is provided with all reagents necessary to run 200 (or 1000) tests or microwell 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 1-2 X 105 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.
          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.
          6. If non-adherent cells are being used, centrifuge 96 well dish for 10 min at 1000 rpm.
          7. Remove contents of wells by inverting over sink and blot gently on paper towels.
          8. Add 200 µl of the Fixative/Denaturing Solution to each well.
          9. Incubate for 30 min at room temperature.
          10. 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.
          11. 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.
          12. 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.
          13. 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.
          14. Prepare the conjugate by diluting the reconstituted (in 1X PBS) 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.
          15. Pipette 100 µl of this solution into each well and incubate for 30 min at room temperature.
          16. 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 Substrate Solution to each well and incubate IN THE DARK at room temperature for 15 min.
          19. Add 100 µl of Stop Solution to each well in the same order as the previously added Substrate Solution.
          20. Measure absorbance in each well using a spectrophotometric plate reader at dual wavelengths of 450-540 nm (or 450-595). Wells must be read within 30 min of adding the Stop Solution.
          Example dataThe promyelocytic cell line HL-60 can be induced to differentiate with phorbol ester (PMA) or retinoic acid (RA). Differentiated HL-60 cells lose the ability to proliferate in culture. To demonstrate the utility of the BrdU Cell Proliferation Assay in measuring the dose response of the two drugs 2 X 10⁴ cells/well (2 X 10⁵ cells/ml) were incubated with various concentrations of PMA or RA for 24 h (37°C, 7% CO₂). BrdU Label was added and the cells incubated for an additional 24 h. Incorporated BrdU was detected by the BrdU Cell Proliferation Assay. The concentrations of these two drugs where HL-60 exits the cell cycle concomitant with the onset of myeloid maturation was found by the BrdU Cell Proliferation Assay. In another series of experiments, the BrdU Cell Proliferation Assay detected the dose of thymidine and phorbol ester that caused cell cycle arrest in both adherent cells line and non-adherent cell lines. A kinetic/sensitivity study was performed using the PMA treated HL-60 system. Various concentrations of HL-60 cells were growth arrested by treatment with 20 ng/ml PMA. After 24 h the HL-60 cells were incubated with BrdU Label for 24, 18, 4 or 2 h and incorporated BrdU was detected by the BrdU Cell Proliferation Assay. There was a direct relationship between the signal and number of proliferating cells at all labeling times. With one exception, the signals of proliferating cells were significantly higher than the background control (-BrdU) and blank control (no cells). The 2-h BrdU labeling at 160 cells/well was not significantly higher than the blank control. Similar results were demonstrated with the Daudi cell line and the HT1080 cell line. The sensitivity of the BrdU Cell Proliferation Assay (Cat. No. QIA58) was compared to a BrdU Proliferation Assay of a competitor using two cell lines. The BrdU Cell Proliferation Assay was more sensitive (signal of proliferating cells over no cells) at all BrdU labeling times. In many different systems both the 24 h and 2 h BrdU labeling times detected proliferating cells.
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