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QIA128 Ultrasensitive Cell Proliferation Assay Kit, Fluorogenic

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
QIA128
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      Overview

      This product has been discontinued.





      A highly sensitive, reliable cell proliferation assay utilizing Calcein-AM, a fluorescent probe for staining viable cells (Ex.: ~485 nm; Em.: ~520 nm). For optimal results serum-free media or Dulbecco’s PBS is recommended. Endogenous esterases in serum can lead to increased blank values which can be subtracted out. Extremely useful for cytotoxicity assays.
      Catalogue NumberQIA128
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Pipettors with disposable tips
      • Tissue culture grade, flat-bottom 96-well black plate
      • Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set of ~485/520 nm
      References
      ReferencesYang, A., et al. 2002. Cell Biol. Toxicol. 18, 97.
      De Clerk, L.S. 1994. J. Immunol. Methods 172, 115.
      Bozyczko-Coyne, D., et al. 1993. J. Neurosci. Methods 50, 205
      Wang, X.M., et al. 1993. Hum. Immunol. 37, 264.
      Product Information
      Detection methodFluorometric
      Form500 Tests
      Format96-well plate
      Kit containsCalcein-AM, Dulbecco-PBS.
      Applications
      Biological Information
      Assay range250-25,000 cells
      Assay time1 h
      Sample TypeViable cells
      Physicochemical Information
      Sensitivity100 Cells
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® Ultrasensitive Cell Proliferation Assay Kit is designed to fluorometrically determine the number of viable cells in proliferation, or for cytotoxicity assays that require greater sensitivity than absorbance-based proliferation assays. The amount of fluorescent dye produced by cellular esterases is proportional to the number of viable cells. The detection range for the number of viable cells is from 100 to 30,000 cells. Due to this high sensitivity the Calbiochem Ultrasensitive Cell Proliferation Assay Kit is also suitable for staining cells in cell-based studies such as cell adhesion or chemotaxis assays.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsUpon arrival store the entire contents of the kit at -20°C.
      Protect from Light Protect from light
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCalcein-AM, Dulbecco-PBS.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA128 0

      Documentation

      Ultrasensitive Cell Proliferation Assay Kit, Fluorogenic Certificates of Analysis

      TitleLot Number
      QIA128

      References

      Reference overview
      Yang, A., et al. 2002. Cell Biol. Toxicol. 18, 97.
      De Clerk, L.S. 1994. J. Immunol. Methods 172, 115.
      Bozyczko-Coyne, D., et al. 1993. J. Neurosci. Methods 50, 205
      Wang, X.M., et al. 1993. Hum. Immunol. 37, 264.
      User Protocol

      Revision19-February-2010 RFH
      Form500 Tests
      Format96-well plate
      Detection methodFluorometric
      StorageUpon arrival store the entire contents of the kit at -20°C.
      Intended useThe Calbiochem® Ultrasensitive Cell Proliferation Assay Kit is designed to fluorometrically determine the number of viable cells in proliferation, or for cytotoxicity assays that require greater sensitivity than absorbance-based proliferation assays. The amount of fluorescent dye produced by cellular esterases is proportional to the number of viable cells. The detection range for the number of viable cells is from 100 to 30,000 cells. Due to this high sensitivity the Calbiochem Ultrasensitive Cell Proliferation Assay Kit is also suitable for staining cells in cell-based studies such as cell adhesion or chemotaxis assays.
      BackgroundCell proliferation and the assessment of substances that either promote or inhibit cell proliferation are crucial to the study of cell biology and to drug-discovery research. The Ultrasensitive Cell Proliferation Assay Kit provides a simple, rapid, and very sensitive procedure for the quantification of viable cells. The assay is suitable for cell proliferation studies as well as for routine cell counts. This assay may also prove useful for the quantification of cells in cell migration or cell adhesion studies. It may be scaled up to 1 ml (e.g. 0.9 ml cell culture and 0.1 ml Calcein-AM working solution) for detection in standard cuvettes. The sensitivity of the assay is 100 cells/well after a 1-h incubation at 37° in a CO2 incubator.
      Principles of the assayThe Calbiochem® Ultrasensitive Cell Proliferation Assay Kit provides a sensitive fluorometric assay for the fast and convenient determination of viable cell numbers in cell proliferation or cell cytotoxicity studies and in cell-based assays such as cell adhesion or cell migration assays. After dilution of the Calcein-AM stock solution with D-PBS, the staining solution is ready to use. It is added to the wells of a 96-well tissue culture plate containing cultivated cells. The intensity of the dye is proportional to the number of viable cells, and the fluorescence is read with a fluorescence microplate reader with excitation at 485 ± 10 nm and emission detection at 520 ± 10 nm after a 1-h incubation at 37°C in a CO₂ incubator.
      Materials provided• Calcein-AM (Kit Component No. JA7705): 4 x 50 µl, sufficient for 500 tests, supplied in anhydrous DMSO in amber glass bottles
      • PBS (Kit Component No. JA9369): 10 ml, supplied in a plastic bottle
      Materials Required but not provided• Pipettors with disposable tips
      • Tissue culture grade, flat-bottom 96-well black plate
      • Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set of ~485/520 nm
      Precautions and recommendationsThe assay should be performed with cells cultured in serum-free media or Dulbecco-PBS. If serum is required use only 1-2% serum as serum components will give rise to higher background values that must be subtracted from the sample values. Figure 3 shows the absorbance of U937 cells cultured in various media/buffer systems. If cells must be cultured in the presence of 10% serum (standard cell culture conditions) replace the cell culture solution with serum-free media or PBS prior to adding the cell staining solution.
      Reagent preparationWarm the Calcein-AM solution and the PBS to room temperature. Dilute the Calcein-AM stock solution 1:30 with PBS. (e.g. 580 µl PBS and 20 µl of Calcein-AM for six 8-well strips of a 96-well plate). For long-term storage the Calcein-AM stock solution should be aliquoted into smaller volumes and tightly sealed to prevent the entry of moisture; store at -20°C and protect from light. The Calcein-AM working solution cannot be stored. Use 10 µl working solution for 100 µl cell culture solution.
      Detailed protocolThe assay should be performed with cells cultured in serum-free media or Dulbecco-PBS. If serum is required use only 1-2% serum as serum components will give rise to higher background values that must be subtracted from the sample values. Figure 3 shows the absorbance of U937 cells cultured in various media/buffer systems. If cells must be cultured in the presence of 10% serum (standard cell culture conditions) replace the cell culture solution with serum-free media or PBS prior to adding the cell staining solution.

      1. Grow cells in a tissue culture grade, flat-bottom plate in 100 µl of serum-free medium or D-PBS per well in a CO2 tissue culture incubator. For cell stimulation assays a starting concentration of 2,500-10,000 cells per well is recommended, for cytotoxicity assays the recommended number of cells per well is 10,000-50,000.

      2. After incubation add 10 µl Calcein-AM working solution, prepared as above, to each well.

      3. Incubate for 1-1.5 h at 37°C in a CO2 incubator.

      4. Measure the fluorescence of the samples using a fluorescence plate reader at excitation 485 ± 10 nm and emission 520 ± 10 nm.
      Example data

      Figure 1: Quantification of HL-60 Cells

      Quantification of HL-60 cells using the Ultrasensitive Cell Proliferation Assay Kit. Fluorescence was measured in a microplate reader with excitation at 485 nm and emission at 520 nm. Cells were incubated with the Calcein-AM working solution for 1 h at 37°C in a CO2 incubator.

      Figure 2: U251 Cells

      Various cell numbers of the adherent cell line U251 were incubated in EMEM containing 1% serum, overnight, at 37°C in a CO2 incubator. After incubation, 10 µl of the working solution was added and incubated for 1 h at 37°C in a CO2 incubator.

      Figure 3: U937 Cells

      Various numbers of suspension cells were cultured in Dulbecco-PBS (♦), in serum-free RPMI media (⊄), or in RPMI media containing 2% serum (Δ) for 6 h at 37°C in a CO2 incubator. This was followed by the addition of 10 µl working solution to each well and incubation for 1 h at 37°C in a CO2 incubator.

      Figure 4: U937 Cells

      20,000 U937 cells per well were incubated overnight at 37°C in the presence of 0 or 10 µg/ml camptothecin in RPMI medium containing 2% FBS. After the incubation 10 µl of the Calcein-AM working solution was added and the absorbance was read after a 1-h incubation at 37°C in a CO2 incubator.
      Sensitivity100 Cells
      Assay Range250-25,000 cells
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.