Propidium iodide
(Synonyms: 碘化丙啶; PI) 目录号 : GC14228
A fluorescent probe used to identify dead cells
Cas No.:25535-16-4
Sample solution is provided at 25 µL, 10mM.
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2 mL of PBS solution containing 0.1 vol% Calcein AM and 0.1 vol% PI (GLPBIO) was added into the samples and incubated for 30 min at 37 °C.
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Quality Control & SDS
- View current batch:
- Purity: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
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.
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]。
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.
| Cas No. | 25535-16-4 | SDF | |
| 别名 | 碘化丙啶; PI | ||
| 化学名 | 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-] | ||
| 分子式 | C27H34I2N4 | 分子量 | 668.39 |
| 溶解度 | 100 mg/mL in DMSO (Need ultrasonic); 5mg/mL in Water (Need ultrasonic). | 储存条件 | Store at -20°C,protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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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 |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry
Cold Spring Harb Protoc2016 Nov 1;2016(11).PMID: 27803250DOI: 10.1101/pdb.prot087288
The surface of healthy cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflet of the plasma membrane. One of these lipids, phosphatidylserine (PS), is normally restricted to the inner leaflet of the plasma membrane and is, therefore, only exposed to the cell cytoplasm. However, during apoptosis lipid asymmetry is lost and PS becomes exposed on the outer leaflet of the plasma membrane. Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane. However, apoptotic cells can be distinguished from necrotic cells by co-staining with propidium iodide (PI) because PI enters necrotic cells but is excluded from apoptotic cells. This protocol describes Annexin V binding and PI uptake followed by flow cytometry to detect and quantify apoptotic and necrotic cells.
Analysis of apoptosis by propidium iodide staining and flow cytometry
Nat Protoc2006;1(3):1458-61.PMID: 17406435DOI: 10.1038/nprot.2006.238
Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.
Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death
J Vis Exp2011 Apr 24;(50):2597.PMID: 21540825DOI: 10.3791/2597
Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability. The Annexin V/PI protocol is a commonly used approach for studying apoptotic cells. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).
Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry
Cold Spring Harb Protoc2016 Jul 1;2016(7).PMID: 27371595DOI: 10.1101/pdb.prot087163
Propidium iodide (PI) is a small fluorescent molecule that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane. PI uptake versus exclusion can be used to discriminate dead cells, in which plasma membranes become permeable regardless of the mechanism of death, from live cells with intact membranes. PI is excited by wavelengths between 400 and 600 nm and emits light between 600 and 700 nm, and is therefore compatible with lasers and photodetectors commonly available in flow cytometers. This protocol for PI staining can be used to quantitate cell death in most modern research facilities and universities.
An Annexin V-FITC-Propidium Iodide-Based Method for Detecting Apoptosis in a Non-Small Cell Lung Cancer Cell Line
Methods Mol Biol2021;2279:213-223.PMID: 33683697DOI: 10.1007/978-1-0716-1278-1_17
Annexin V and propidium iodide staining is widely used for determining the cellular death through apoptosis. In the presence of Ca2+ ions, annexin V has a strong binding affinity for phosphatidylserine, a membrane phospholipid that during apoptosis is translocated from the inner side of the cell membrane to its outer side. On the other hand, propidium iodide has ability for DNA binding and it can only enter into necrotic or late apoptotic cells. This chapter describes a commonly used method for detection of apoptosis in a non-small cell lung cancer cell line using annexin V and propidium iodide dye. We describe the detection of different stages of apoptosis in the A549 lung cancer cell line treated with dihydroartemisinin (DHA). This apoptosis detection method can be used to determine the efficacy of different kinds of drugs on cultured cancer cell lines.