Propidium iodide

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.

CRITICAL：Prepare fresh staining solution just before use.

1. Suspend cells at 1–2 × 10⁶ cells ml⁻¹ 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.