Nuclear staining agent
Nucleic acids (intercalates with DNA/RNA with a stoichiometry of one dye per 4-5 base pairs, little sequence preference) [1]

Guidelines for use:
1.1 Preparation of stock solution: prepare 1 mg/mL stock solution in ddH2O.
1.2 Preparation of working solution: Dilute the Propidium Iodide stock solution to a working solution of 20–50 μg/mL using either preheated serum-free cell culture medium or PBS.
Note: Before use, please make sure that the concentration of the propidium iodide working solution is appropriate for your specific needes, you may adjust the concentration ofworking solution and incubation period.

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2. Cell staining (suspension cells)
2.1 Centrifuge the cells, add PBS, and wash twice for a total of five minutes. One x 10^6 cells per milliliter.
2.2 After adding 1 mL of working solution, let it sit at room temperature for five to ten minutes.
2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant.
2.4 Wash the cells twice, for five minutes each time, with PBS added.
2.5 After resuspending cells in 1 mL serum-free medium or PBS, observe using a fluorescence microscope or flow cytometer.

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3. Cell staining (adherent cells)
3.1 On clean coverslips, cultivate the adhering cells.
3.2 Take out the coverslip and remove any extra culture medium.
3.3 Add 100 μL of the Propidium Iodide working solution, give the cells a brief shake to fully coat them, and let them sit for five to thirty minutes. Propidium Iodide. 3.4 Rinse with culture media after aspirating out the dye working solution. Use a fluorescent microscope to observe two to three times, for five minutes at a time.
Note: If flow cytometry is to be used, the cells must first be digested by trypsin and resuspended before being stained.

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Storage conditions: -20°C, protect from light and being moisturized, 1 year.

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Safety notes:
1. Depending on the experimental needs, you may adjust the concentration of propidium iodide working solution and incubation period.
2. This product is intended solely for use in scientific research. It is prohibited from usage in food or medication, as well as in clinical diagnosis or treatment.
3. Propidium Iodide can cause cancer.Please wear a lab coat and disposable gloves for your health and safety.

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Pre‑fixation PI staining (applied 2 h before fixation) in NMDA‑lesioned organotypic hippocampal slice cultures (OHSC) resulted in brightly labeled condensed (pycnotic) nuclei of degenerating neurons, while viable cells remained unstained. [1]
- Post‑fixation PI staining (applied after fixation) in the same lesioned OHSC labeled both pycnotic nuclei and cell bodies of non‑degenerating viable cells, revealing the entire tissue cytoarchitecture. [1]
- In brain cryostat sections, post‑fixation PI staining (5 μg/mL, 2 h) produced a diffuse labeling of neuronal perikarya with bright nucleoli but nearly unstained nuclei; in contrast, glial cells showed intense nuclear PI labeling with virtually unlabeled cell bodies. [1]
- PI staining pattern after post‑fixation was highly consistent with hemalaun staining in bright field microscopy, allowing visualization of cellular stratification, glial cell arrangement, and vessel architecture. [1]
- Post‑fixation PI staining combined with immunofluorescence (NeuN, calbindin, calretinin, GFAP) or lectin (IB4) enabled simultaneous visualization of cytoarchitecture and specific cell markers in postnatal (p1, p9) and adult rat brain sections. [1]
- In early postnatal tissues (p1), conventional markers (NeuN, calbindin) showed incomplete staining, whereas post‑fixation PI staining fully revealed cytoarchitecture independent of developmental stage. [1]

Flow cytometric analysis: Propidium iodide is prepared in 0.1% sodium citrate + 0.1% Triton X-100 (50 μg/mL). Then the 200 ×g centrifuged cell pellet is gently resuspended in 1.5 mL hypotonlc fluorochrome solution (Propidium iodide 50 μg/mL), in 12×75 polypropylene tubes. The tubes are placed at 4°C in the dark overnight before the flow cytometric analysis. The propidium Iodide fluorescence of individual nuclei is measured using a FACScan flow cytometer. The nuclei traverses the light beam of a 488 nm Argon laser. A 560 nm dichrolc nurror (DM 570) and a 600 nm band pass filter (bandwidth 35 nm) are used for collecting the red fluorescence due to propidium Iodide staining of DNA and the data are registered on a logarithmic scale. [2]

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Propidium Iodide is a red fluorescent used to stain cells. Propidium Iodide only can be taken up by cells with impaired membrane integrity, it can be used in cell cycle and cell viability assays, as well necrocytosis assays.

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Example 1, the method for cell nucleus staining is shown below:
1. Cells are washed with PBS, and then incubates cells with Propidium Iodide (15 min).
2. Fluorescent images are acquired with a confocal laser scanning microscope.

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Example 2, the method for cell staining is shown below:
1. Fix cultured cells with paraformaldehyde (4%; 10 min).
2. Incubate cells with Propidium Iodide (50 mg/L; 15 min; dark).
3. Fluorescent images are acquired with a confocal laser scanning microscope.

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Pre‑fixation PI staining for degenerating neurons: Organotypic hippocampal slice cultures (OHSC) were lesioned with 50 μM NMDA for 4 h, then rinsed and cultured for another 3 days. PI (5 μg/mL) was added 2 h before fixation. After washing, cultures were fixed with 4% paraformaldehyde overnight. PI specifically labeled pycnotic nuclei of degenerating neurons in the granule cell layer of the dentate gyrus, while viable cells remained unlabeled. [1]
- Post‑fixation PI staining for cytoarchitecture: OHSC or brain cryostat sections (20, 200, 500, or 1000 μm thickness) were first fixed with 4% paraformaldehyde overnight, then incubated with PI (5 μg/mL) for 2 h. This procedure stained both degenerating and viable cells, revealing cell bodies, nuclei (nucleoli brightly stained, rest of nucleus nearly negative in neurons), and glial cells (intensely stained nuclei). Staining was uniform throughout the entire sample without gradient up to 1 mm thickness. [1]
- Combined PI and immunofluorescence/lectin staining on brain sections: Cryostat sections (20 μm) from postnatal day 1 (p1), p9, and adult rats were post‑fixed, then incubated with primary antibodies (anti‑NeuN, anti‑calbindin, anti‑calretinin, anti‑GFAP) or FITC‑conjugated IB4, followed by secondary antibodies. PI staining (5 μg/mL, 2 h) was performed either before or after antibody incubation. PI allowed assessment of total cytoarchitecture while specific markers showed developmental expression patterns (e.g., NeuN faint at p1, increasing by p9 and adult; calbindin expression followed outside‑in migrational gradient; GFAP increased with maturation). [1]
- Counterstaining with Sytox green: OHSC subjected to post‑fixation PI staining were counterstained with Sytox green (1:10000 dilution, 5 min). Sytox green labeled all nuclei, while PI provided additional cytomorphological details (cell size, cell allocations). Overlap of PI and Sytox green showed yellow matches. [1]

Brain tissue preparation: Wistar rats of different postnatal developmental stages (p1, p9, and adult) were deeply anesthetized with isoflurane and sacrificed by decapitation. Brains were removed and immediately fixed by immersion in 4% paraformaldehyde in 0.2 M phosphate buffered saline overnight. Brains were then cryoprotected with ascending 10%, 20%, and 30% sucrose solutions, cut in the transversal plane on a cryostat (20 μm thickness, as well as 200, 500, and 1000 μm sections), and mounted on gelatine‑coated slides. No drug administration or in vivo treatment with Propidium Iodide was performed. [1]

[1]. Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain. Micron. 2012 Oct;43(10):1031-8.

[2]. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991 Jun 3;139(2):271-9.

Propidium iodide is an organic iodide salt containing propidium iodide. Propidium iodide is a fluorescent nucleic acid dye that binds only to double-stranded nucleic acids. Its molecular weight is 668.4, with a maximum absorption wavelength of 535 nm and a maximum emission wavelength of 617 nm. It is commonly used to determine the DNA content of cells or to distinguish between live and dead cells. Propidium iodide is a quaternary ammonium salt analog of ethidium; it is an intercalating dye with specific affinity for certain types of DNA and is used for density gradient separation in the form of diiodide; it can also form a fluorescent complex with cholinesterase and inhibit the activity of this enzyme. Immunohistochemistry has become an indispensable technique in biomedical research, used to study cell morphology and pathology under bright-field and fluorescence microscopy. Although some staining methods, such as Nissl staining or hematoxylin-eosin staining, can reveal complete cell structures under bright-field microscopy, there are still limitations in observing cell structures under fluorescence microscopy. This study reports a simple staining method using the fluorescent dye propidium iodide (PI, 5 μg/mL) to clearly visualize cell distribution and composition in the central nervous system of adult and developing rodents under fluorescence microscopy. PI is a recognized marker of degenerated cells and is usually used before fixation (prefixation PI staining). In this study, PI was added to the sections after fixation (postfixation PI staining). This improved labeling method yielded a similar staining pattern of cell structures under fluorescence microscopy to that observed under bright-field microscopy with hematoxylin staining. This finding has been validated in organotype hippocampal culture (OHSC) and brain sections from different postnatal developmental stages. Prefixation PI staining in excitotoxically damaged OHSCs only revealed brightly labeled condensed nuclei of degenerated neurons. In contrast, postfixation PI staining further revealed extensive labeling of neuronal cell bodies and glial cells within OHSCs, allowing observation of the layered structure and cell morphology of neuronal layers. In addition, in postnatal (p1 and p9) and adult rats, fixed PI staining was used in combination with NeuN, calcium-binding protein, calreticulin, glial fibrillary acidic protein, or Griffonia simplicifolia heterolectin B4 (IB(4)). In early postnatal brain slices, almost all of the above-mentioned cell markers resulted in incomplete staining of native cell structures, leading to inaccurate assessment of cell morphology compared to the adult brain. In contrast, fixed PI staining allows for the study of whole cell structures independently of developmental stages. In summary, fixed PI staining provides detailed information on the morphology of developing and adult brain tissues under a fluorescence microscope. [1]

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Corticosteroids, calcium ionophores, and anti-CD3 monoclonal antibodies kill cultured mouse thymocytes in vitro. Extensive DNA fragmentation occurs prior to cell death, forming oligonucleotide subunits. This cell death (apoptosis) occurs physiologically during the selection of immune cells in the thymus and is usually assessed by electrophoresis or colorimetry, which measures the degree of DNA fragmentation in nuclear extracts. However, these techniques cannot determine the percentage of apoptotic cell nuclei or identify apoptotic cells within heterogeneous cell populations. We developed a flow cytometry method to measure the percentage of apoptotic cell nuclei after staining with propidium iodide in hypotonic buffer and compared it with classic colorimetric and electrophoretic methods using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic cell nuclei appeared as a broad, low-diploid DNA peak in the red fluorescent channel, easily distinguishable from the narrow peak of thymocytes with normal (diploid) DNA content. No low-diploid DNA peak was observed when DEX-induced apoptosis was inhibited by incubation at low temperature (4°C) or treatment with cyclohexylimide. Similarly, thymocyte death induced by sodium azide (a substance that kills cells through non-apoptotic mechanisms) did not result in any change to the normal DNA peak. Flow cytometry data showed excellent correlation with electrophoretic and colorimetric results. This novel, rapid, simple, and reproducible method should be helpful in assessing apoptosis in specific cell populations in heterogeneous tissues such as bone marrow, thymus, and lymph nodes. [2]

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Propidium iodide is a cell‑membrane impermeable fluorophore with excitation maximum at 535 nm and emission maximum at 617 nm. It intercalates with nucleic acids with a stoichiometry of one dye per 4‑5 base pairs and little sequence preference. [1]
- Pre‑fixation PI staining (applied before fixation) is widely used for quantitative assessment of neuronal cell decline in models of acute neurodegeneration, visualized as intensely labeled PI‑positive pycnotic nuclei of degenerating neurons. [1]
- Post‑fixation PI staining (applied after fixation) allows dye diffusion into originally intact cells because tissue fixation with paraformaldehyde disrupts cell membrane integrity, resulting in consistent cytoarchitectural staining. This method provides an equivalent in fluorescence microscopy to hematoxylin‑based staining (e.g., hemalaun) in bright field microscopy. [1]
- Post‑fixation PI staining can be combined with multiple immunolabeling techniques (NeuN, calbindin, calretinin, GFAP) and lectin staining (IB4). [1]
- The method is particularly useful for studying early postnatal brain tissues because common immunolabeling markers often show incomplete expression in immature tissues, whereas PI reveals full cytoarchitecture independent of developmental stage. [1]
- Tissue penetration capacity: consistent staining without gradient was observed in sections up to 1000 μm thickness. [1]

C27H34I2N4

668.41

668.087

C, 48.52 H, 5.13 I, 37.97 N, 8.38

25535-16-4

104981

Pink to red solid powder

220-225 °C (dec.)(lit.)

0.158

2

4

7

33

555

0

[I-].[I-].[N+](C([H])([H])[H])(C([H])([H])C([H])([H])[H])(C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])[N+]1=C2C([H])=C(C([H])=C([H])C2=C2C([H])=C([H])C(=C([H])C2=C1C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])[H])N([H])[H]

XJMOSONTPMZWPB-UHFFFAOYSA-M

InChI=1S/C27H33N4.2HI/c1-4-31(3,5-2)17-9-16-30-26-19-22(29)13-15-24(26)23-14-12-21(28)18-25(23)27(30)20-10-7-6-8-11-20;;/h6-8,10-15,18-19,29H,4-5,9,16-17,28H2,1-3H3;2*1H/q+1;;/p-1

3-(3,8-Diamino-6-phenylphenanthridin-5-ium-5-yl)propyl-diethyl-methylazanium diiodide

Propidium Diodide Propidium Iodide; Propidium diiodide; 3,8-Diamino-5-(3-(diethyl(methyl)ammonio)propyl)-6-phenylphenanthridin-5-ium iodide; Propidium Iodine; Propidium (Iodide); CHEBI:51240; 3,8-Diamino-5-(3-(diethylmethylammonio)propyl)-6-phenylphenanthridinium diiodide; ...

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~149.61 mM)
H2O : ~3.57 mg/mL (~5.34 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.74 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: Solubility in Formulation 1: ≥ 2.5 mg/mL (3.7 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 25 mg/mL DMSO stock solution and add to 400 μL of PEG300, mix well (clear solution); Then add 50 μL of Tween 80 to the above solution, mix well (clear solution); Finally, add 450 μL of saline to the above solution, mix well (clear solution).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.7 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 25 mg/mL DMSO stock solution and add to 900 μL of 20% SBE-β-CD in saline, mix well (clear solution).
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)