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Propidium iodide (Synonyms: PI)

Catalog No.GC14228

Propidium iodide (PI) is a small red-fluorescent molecule that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane.

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Propidium iodide Chemical Structure

Cas No.: 25535-16-4

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500mg
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Sample solution is provided at 25 µL, 10mM.

Product has been cited by 2 publications

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Protocol

Procedure for Propidium iodide staining [1]

Fluorochrome solution:  0.1% sodium citrate (wt/v), 0.1% Triton X-100 (v/v), 50 mg l−1 Propidium iodide in deionized/distilled water). Fluorochrome solution can be kept in the dark at 4 °C for months.

DNA staining solution:  Dissolve 200 μg of Propidium iodide in 10 ml of PBS. Add 2 mg of DNase free RNase.

CRITICALPrepare fresh staining solution just before use.

  1. Suspend cells at 1–2 × 106 cells ml1 in 1 ml of PBS in 12×75 tubes.
  2. Centrifuge at 200g for 5 min at room temperature.
  3. Aspirate off the PBS.
  4. Follow the quick method for thymocytes and non-adherent mononuclear cells (option A) and the standard method for multinuclear cells growing in suspension and for adherent cells (option B).
  5. Quick method (direct DNA staining in PI hypotonic solution)

i)  Gently resuspend the cell pellet in 1 ml of fluorochrome solution.

ii)  Place the tubes in the dark at 4 °C, before flow cytometry analysis, for at least 1 h and no longer than 24 h.

Note: One hour is necessary for appropriate staining of the nuclei; cells can be maintained for 24 h, in the dark at 4 °C without any substantial change in DNA profile.

B.  Standard method (PI staining after alcoholic fixation)

i)  Resuspend cell pellet in 500 μl of PBS.

ii)  Fix cells by adding 4.5 ml of 70% (v/v) cold ethanol to the cell suspension keeping the tubes on ice.

PAUSE POINT: Cells can be stored in ethanol solution at −20 °C for several weeks.

iii)  Centrifuge at 400g for 5 min and remove the supernatant (ethanol solution).

iv)  Wash cells in 5 ml of PBS and centrifuge at 400g for 5 min.

CRITICAL STEP: Cells with extensive DNA degradation can be directly resuspended in DNA staining solution without any further treatment.

  v)  If DNA is not extensively degraded, resuspend cells in 0.5 ml of PBS and add 0.5 ml of DNA extraction buffer. Incubate at room temperature for 5 min and centrifuge at 400g for 5 min.

vi) Remove the supernatant and resuspend cells in 1 ml of DNA staining solution.

  vii) Incubate resuspended cells for at least 30 min at room temperature in the dark.

  1. Analyze cells by flow cytometry. Use 488-nm laser line for excitation. Measure red fluorescence (>600 nm) and side scatter. Collect at least 20,000 events. Gate-out residual debris. Measure hypodiploid and diploid DNA peaks.

 

 

This protocol only provides a guideline, and should be modified according to your specific needs

 

[1]. Riccardi C, Nicoletti I. Analysis of apoptosis by propidium iodide staining and flow cytometry[J]. Nature protocols, 2006, 1(3): 1458-1461.

Background

Propidium iodide (PI) is a small red-fluorescent molecule that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane. Propidium iodide uptake versus exclusion can be used to discriminate dead cells. PI is excited by wavelengths between 400 and 600 nm and emits light between 600 and 700 nm [1]. PI intercalates to DNA with no sequence preference with one dye molecule per four to five base pairs [2]. When bound to DNA fluorescence of PI is enhanced 20- to 30-fold [3]. PI binds stoichiometrically to nucleic acids so that fluorescence emission is proportional to the DNA (and RNA, which has to be removed if DNA is to be measured) content of a cell [4].

References:
[1]. Crowley L C, Scott A P, Marfell B J, et al. Measuring cell death by propidium iodide uptake and flow cytometry[J]. Cold Spring Harbor Protocols, 2016, 2016(7): pdb. prot087163.
[2]. Waring M J. Complex formation between ethidium bromide and nucleic acids[J]. Journal of molecular biology, 1965, 13(1): 269-282.
[3]. Arndt-Jovin D J, Jovin T M. Fluorescence labeling and microscopy of DNA[J]. Methods in cell biology, 1989, 30: 417-448.
[4]. Riccardi C, Nicoletti I. Analysis of apoptosis by propidium iodide staining and flow cytometry[J]. Nature protocols, 2006, 1(3): 1458-1461.

Chemical Properties

Cas No. 25535-16-4 SDF
Synonyms PI
Chemical Name 3,8-diamino-5-(3-(diethyl(methyl)ammonio)propyl)-6-phenylphenanthridin-5-ium iodide
Canonical SMILES NC1=CC2=[N+](C(C3=CC=CC=C3)=C(C=C4N)C(C=C4)=C2C=C1)CCC[N+](CC)(C)CC.[I-].[I-]
Formula C27H34I2N4 M.Wt 668.39
Solubility 100 mg/mL in DMSO (Need ultrasonic); 5mg/mL in Water (Need ultrasonic). Storage Store at 2-8°C, protect from light
General tips Please select the appropriate solvent to prepare the stock solution according to the solubility of the product in different solvents; once the solution is prepared, please store it in separate packages to avoid product failure caused by repeated freezing and thawing.Storage method and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored at -20°C, please use it within 1 month.
To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time.
Shipping Condition Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request.

Complete Stock Solution Preparation Table

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1 mg 5 mg 10 mg
1 mM 1.4961 mL 7.4807 mL 14.9613 mL
5 mM 0.2992 mL 1.4961 mL 2.9923 mL
10 mM 0.1496 mL 0.7481 mL 1.4961 mL
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