Nuclear staining agent

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.

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]

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.

[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. It contains a propidium.
Propidium Iodide is a fluorescent nucleic acid dye which binds only to double-stranded nucleic acids. It has a molecular weight of 668.4, an absorbance maximum of 535nm, and an emission maximum of 617 nm. It is commonly used to determine the DNA content of a cell or to discriminate viable from non-viable cells.
Quaternary ammonium analog of ethidium; an intercalating dye with a specific affinity to certain forms of DNA and, used as diiodide, to separate them in density gradients; also forms fluorescent complexes with cholinesterase which it inhibits.

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Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell morphology when compared to adult brains. In contrast, post-fixation PI staining allowed investigation of the whole cytoarchitecture independent of the developmental stage. Taken together, post-fixation PI staining provides a detailed insight in the morphology of both developing and adult brain tissues in fluorescence microscopy.[1]

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Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4 degrees C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes. [2]

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

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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.)