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CellSimple™ Propidium Iodide (PI)/RNase Staining Solution Kit Protocol

A. Solutions and Reagents

  1. Propidium Iodide (PI) / RNase Staining Solution (#4087).
  2. 10% Triton™ Cell Lysis Buffer (#14050).

Additional Reagents (Not Supplied)

  1. Phosphate Buffered Saline (PBS-20X) (#9808).

B. Instrumentation

  1. CellSimple™ Cell Analyzer #88416
  2. CellSimple™ Cassettes #35423

C. Reagent Preparation

NOTE: Allow all reagents to reach room temperature.

  1. 1X PBS: To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.

NOTE: For flow cytometry application, adding 0.5% BSA to 1X PBS buffer may help prevent cell loss.

D. Cell Cycle Assay

  1. Prepare a volume of cells ranging from 0.25 – 1.0 ml and a density of 2.5 x 105 to 5 x 105 cell/ml.
  2. Centrifuge cells at 300 G for 5 min then aspirate media.
  3. Wash cells using 1.0 ml of 1X PBS, repeat step 2.
  4. Aspirate 1X PBS and re-suspend cells in PI/RNase staining solution (same volume used in step 1).
  5. If using live cells 10% Triton™ Cell Lysis Buffer needs to be added to permeabilized the cell membrane. A final concentration of 0.05% or a 1:200 dilution from the 10% stock solution will be adequate for this step.
  6. Incubate cells at 37°C for 15 min then analyze using the Cell Cycle App on the CellSimple™ Cell Analyzer.

NOTE: Cells may need to be diluted for optimal data analysis, use the PI/RNase Staining Solution as a diluent.

posted April 2016

Product Includes Quantity (with Count)
Propidium Iodide (PI)/RNase Staining Solution 4087 1 x 25 ml
10% Triton™ Cell Lysis Buffer 1 x 10 ml

Product Description

CellSimple™ Propidium Iodide (PI)/RNase Staining Solution Kit is designed for use with the CellSimple™ Cell Analyzer. It detects cellular DNA content in both living and fixed cells. The median fluorescent intensity (MFI) of the red emission (PMT1) can be used as an indicator for cellular DNA content. This kit can be used together with Cell Cycle App on the CellSimple™ Cell Analyzer for cell cycle progression analysis. At the recommended reagent concentrations and volumes, one kit will provide for 100 assays. The CellSimple™ Cell Analyzer comes complete with a pack of 25 cassettes (50 tests), a USB cord and power adapter, and user documentation.

The CellSimple™ Cell Analyzer is currently only available in the US.

Product Usage Information

As a last step for fluorescent assays, add enough neat (undiluted) CellSimple™ Propidium Iodide (PI)/RNase Staining Solution to submerge cells or samples. Incubate for 15 minutes at room temperature, protected from light before analysis

Storage: Store at 4°C. Protect from light. Kit components are stable for 12 months.

Specificity / Sensitivity

Species Reactivity: Human, Mouse, Rat
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.

Propidium Iodide (PI) is a fluorescent dye which intercalates between bases and stains both DNA and RNA. Specific DNA staining is achieved by enzymatic removal of RNA with a ribonuclease (RNase). PI/RNase is commonly used as a nuclear stain in fluorescent microscopy and as a DNA content determinant in cell cycle analyses by flow cytometry. Cells in G2 and M phases of the cell cycle contain twice the DNA content compared to those in G0 and G1 phases. DNA content during S phase lies between these extremes. PI/RNase Staining Solution has excitation and emission maxima of 535 and 617 nm, respectively (orange to red range of the spectrum).

1.  Suzuki, T. et al. (1997) J Histochem Cytochem 45, 49-53.

2.  Jones, K.H. and Kniss, D.A. (1987) J Histochem Cytochem 35, 123-5.

3.  Deitch, A.D. et al. (1982) J Histochem Cytochem 30, 967-72.

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.
Triton is a trademark of The Dow Chemical Company.

CellSimple™ Propidium Iodide (PI)/RNase Staining Solution Kit