Propidium iodide|25535-16-4|安捷凯

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].

碘化丙锭 (PI) 是一种红色荧光小分子，可与 DNA 结合，但不能被动进入具有完整质膜的细胞。碘化丙锭摄取与排除可用于区分死细胞。 PI 被 400 至 600 nm 之间的波长激发并发射 600 至 700 nm 之间的光^[1]。 PI 以每 4 到 5 个碱基对一个染料分子插入到没有序列偏好的 DNA 中^[2]。当与 DNA 结合时，PI 的荧光增强了 20 到 30 倍^[3]。 PI 以化学计量方式与核酸结合，因此荧光发射与细胞的 DNA（和 RNA，如果要测量 DNA 则必须去除）含量成正比^[4]。

参考文献:
[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.

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.

CRITICAL：Prepare fresh staining solution just before use.

Suspend cells at 1–2 × 10⁶ cells ml^?¹ in 1 ml of PBS in 12×75 tubes.
Centrifuge at 200g for 5 min at room temperature.
Aspirate off the PBS.
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).
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.

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.