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Protocol for CellSimple™ Cell Health Assay Kit #83323 and Cell Health Assay Kit

A. Instrumentation:

The CellSimple™ Cell Analyzer Kit was specially designed for use with the CellSimple™ Cell Analyzer. However, either kit may be used with a flow cytometer or plate reader capable of providing excitation between 480 nm and 490 nm and detecting fluorescent emission between 520 nm and 590 nm.

B. Kit components:

  • Calcein-AM
  • Propidium Iodide (PI Solution)
  • Additional component included with #83323
  • Phosphate Buffered Saline (PBS-20X)

    Note: Calcein-AM stock solution in DMSO is stable for up to 12 months at -20ºC if protected from light and moisture. We recommend aliquoting the stock solution. Close cap tighly after each use. Any aqueous dilution of the Calcein-AM stock should be used within the same day.

C. Additional reagents needed, but not supplied.

  • DMSO
  • Reverse osmosis/deionized (RO/DI) water or equivalent

D. Reagent preparation

NOTE: Allow all reagents to reach room temperature before use.

  1. 1X PBS: To prepare 1 L 1X PBS add 50 ml PBS-20X to 950 ml RO/DI water, mix. Note: For flow cytometry application, adding 0.5% BSA to 1X PBS buffer may help to prevent cell loss.
  2. 2 mM Calcein-AM Solution: Add 20 µl DMSO to each vial of Calcein-AM to make a 2 mM stock solution. Each vial contains enough 2 mM Calcein-AM stock solution for 100 CellSimple™ assays, 200 flow cytometry assays or 2 x 96-well plates.
  3. 150 µM Propidium Iodide
  4. Notes:

    • Calcein-AM solution should be used within 2 months after reconstitution and should be stored desiccated at -20°C protected from light.
    • For optimal labeling in different cell lines, a titration of both Calcein-AM and PI at a final concentration between 0.1 to 10 µM is recommended for both live and dead cells. Treatment of cells with 0.05% Triton™ X-100 or 0.05% digitonin can be used to generate dead cells with damaged plasma membranes for testing purposes.
    • Calcein-AM stock solution in DMSO is stable for up to 12 months at -20°C if protected from light and moisture. We recommend aliquoting the stock solution. Close cap tighly after each use. Any aqueous dilution of the Calcein-AM stock should be used within the same day.
    1. Labeling Solution for CellSimple™ Cell Analyzer assay: Dilute 1 µl of 2 mM stock Calcein- AM with 199 µl DMSO to make a 10 µM Calcein-AM working solution. For CellSimple™ assays add 2.5 µl of 10 µM Calcein-AM working solution and 1.67 µl of 150 µM Propidium Iodide to each 0.5 ml cell suspension to reach a final concentration of 0.05 µM Calcein-AM and 0.5 µM Propidium Iodide.
    2. Labeling Solution for flow cytometry assay: Dilute 2 µl of 2 mM stock Calcein-AM with 98 µl DMSO to make a 40 µM Calcein-AM working solution. For flow cytometry assays add 1.25 µl of 40 µM Calcein-AM working solution and 20 µl of 150 µM Propidium Iodide to each 1.0 ml cell suspension to reach a final concentration of 0.05 µM Calcein-AM and 3 µM Propidium Iodide.
    3. Labeling Solution for 96-well plate assay: Prepare a 2 µM Calcein-AM, 3 µM Propidium Iodide in 1X PBS labeling solution by adding 10 µl of 2 mM Calcein-AM and 200 µl of 150 µM Propidium Iodide to 10 ml 1X PBS. This will provide enough labeling solution for one 96-well plate assay at the recommended conditions.

E. Cell Health Assay protocol using the CellSimple™ Cell Analyzer

  1. Prepare reagents according to Section D.
  2. Harvest cells by centrifugation.
  3. Wash cells once with 1X PBS and resuspened cells in 0.25 mL of 1X PBS at a concentration of 1 x 105 to 5 x 105 cell/ml.
  4. Note: Adherent cells can be detached with EDTA. For toxicity assays, make sure to collect all dead cells floating in the medium.

  5. Apply CalceinAM to a final concentration of 0.01 - 0.05 µM and Propidium Iodide to a final concentration of 0.5 - 5.0 µM to the cell suspension.
  6. Mix well and incubate cells at room temperature for 15 to 30 min. Protect from light.
  7. Analyze samples with a CellSimple™ Cell Analyzer using the Cell Health Application.

F. Cell Health Assay protocol using flow cytometry

  1. Prepare reagents according to Section D.
  2. Harvest cells by centrifugation.
  3. Wash cells once with 1X PBS and resuspend cells in 1 mL 1X PBS at a concentration of 1 x 105 to 5 x 106 cell/ml.
  4. Note: Adherent cells can be detached with EDTA. For toxicity assays, make sure to collect all dead cells floating in the medium.

  5. Apply Calcein-AM to a final concentration of 0.1 - 10 µM and Propidium Iodide to a final concentration of 0.1 - 10 µM. See part 4 of section D.
  6. Mix well and incubate cells at room temperature for 15 to 30 min. Protect from light.
  7. Analyze samples with a flow cytometer set at excitation/emission of 488/535 nm to detect live cells, and an excitation/emission setting of 488/620 nm to detect dead cells.

G. Cell Health Assay protocol using a plate reader (96-well plate assay)

  1. Prepare reagents according to Section D.
  2. For cells in suspension:
    1. Harvest cells and wash once with 1X PBS. Make a 1 x 105 to 1 x 106 cells/ml cell suspension with 1X PBS. Proceed to Step 4.
  3. For adherent cells: Seed cells into a 96 well plate in warm culture medium and culture cells in incubator overnight to allow cells to attach to plate. Typical cell number is between 1 x 104 to 5 x 104 cells/ well. Note: A cell number titration may be necessary for optimal results.
  4. Remove the medium from the plate and wash cells once with 1X PBS. Add 100 µl/well of 1X PBS to plate followed by treatment with desired growth factors or cytotoxic reagents. Note: Because labeling solution will be added directly to this plate, use an FBS-free cell treatment to avoid Calcein-AM signal loss due to serum esterase.
  5. Add 100 µl/well of Labeling Solution to cell plate and incubate cells at room temperature for 30 to 60 min while protected from light.
  6. Analyze samples on a plate reader or fluorescent microscope set at excitation/ emission of 490/520 nm for live cells, and an excitation/emission setting of 535/620 nm for dead cells.

posted June 2016

Product Includes Quantity (with Count)
Calcein AM 1 x 40 µg
Propidium Iodide (PI) Solution 1 x 200 µl
Phosphate Buffered Saline (PBS-20X) 9808 1 x 25 ml

Product Description

The CellSimple™ Cell Health Assay Kit is a fluorescent assay designed for use with the CellSimple™ Cell Analyzer. The Cell Health Assay can determine cell viability by measuring intracellular esterase activity and plasma membrane integrity. The assay kit contains the fluorescent dyes Calcein-AM and Propidium Iodide (PI) and is therefore able to stain both live and dead cells simultaneously.


Product Usage Information

Storage: All components in this kit are stable for at least 12 months when stored at the recommended temperature and left unused. Upon receipt, #11733 and #9808 should be removed from kit box and stored at 4ºC and room temperature, respectively.

Specificity / Sensitivity


Species Reactivity: Human
Species predicted to react based on 100% sequence homology: All Species Expected

The CellSimple™ Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.


Measures of cell viability and cytotoxicity are broadly used to study the effects of growth factors and cytokines, inhibitors and activators, and immune response signals. Calcein-AM is the acetomethoxyl form of calcein, a highly lipophilic, cell membrane permeable dye. Intracellular esterase activity converts the non-fluorescent Calcein-AM to the highly fluorescent Calcein, which is retained only within live cells (1,2). The DNA-binding agent PI is cell membrane impermeable and only enters dead cells or those with damaged cell membranes. Intracellular PI binds DNA and undergoes an approximate 40-fold enhancement in fluorescence intensity. As a result, live cells will produce a strong green fluorescence resulting from the conversion of Calcein-AM to Calcein, while dead cells produce a strong red fluorescence due to the presence of PI (3,4). These fluorescent signals can be detected using the Cell Health application on the CellSimple™ Cell Analyzer.


1.  Papadopoulos, N.G. et al. (1994) J Immunol Methods 177, 101-11.

2.  Decherchi, P. et al. (1997) J Neurosci Methods 71, 205-13.

3.  Cárdenas, W. et al. (2004) Fish Shellfish Immunol 17, 223-33.

4.  Hiraoka, Y. and Kimbara, K. (2002) Appl Environ Microbiol 68, 2031-5.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
CellSimple is a trademark of Cell Signaling Technology, Inc.

83323
CellSimple™ Cell Health Assay Kit