Dec 21, 2015 viability is a common issue of concern in almost all microbial processes. Critical aspects of using bacterial cell viability assays with the fluorophores syto9 and propidium iodide philipp stiefel, sabrina schmidtemrich, katharina maniuraweber and qun ren abstract background. It is predominantly used to measure fluorescence intensity produced by fluorescentlabeled antibodies detecting proteins, or ligands that bind to specific cellassociated molecules such as propidium iodide binding to dna. Propidium iodide pi is a fluorescent dye that binds to dna. Is there an alternative to propidium iodide for dead cell staining. Fluorescence microscopy protocol for counterstaining cells with propidium iodide 1. The staining procedure involves making a singlecell suspension from cell culture or tissue samples. The fluorescence intensity of rh123 reflects the amount of atp generated in mitochondria. View sample under fluorescence microscope adjusted at appropriate filters. The method used will depend on the experiment and the information required. Am dye to the fluorescent calcein dye by intracellular esterase activity in live cells. As in the case of ethidium bromide its fluorescence is enhanced for 2030fold upon binding to nucleic acids. Harvest cells in the appropriate manner and wash in pbs. Propidium iodide solution is used to assess neuronal death.
Pi binds to dna by intercalating between the bases with little or no sequence preference. Is there an alternative to propidium iodide for dead cell. Staining dead cells with propidium iodide pi or 7aminoactinomycin d 7aad propidium iodide and 7aad can be used to stain dead cells so that they may be excluded from analysis in standard live cell surface staining protocols. For easy setup, with pi staining of dna content for flow cytometry we recommend our propidium iodide flow cytometry kit, otherwise, we recommend this protocol. Bacteria stain propidium iodide solution 1 mgml in h2o. I would like to identifiy necrosis by fluorescence microscopy in my wound. Propidium iodide pi is used as a marker of cell membrane permeability see figure 1 on page 7. The appearance of phosphatidylserine ps residues normally hidden within the plasma membrane on the surface of the cell is an early event in apoptosis, and can be used to detect and measure apoptosis. Flow cytometry protocol for counterstaining suspended cells with propidium iodide 1.
Propidium iodide may be used in flow cytometry to evaluate cell viability when used with other dyes that stain viable cells or cells that are early in the apoptosis process. However, it can easily penetrate dead or damaged cells and as such is commonly used for identifying cell viability in a population or as a counterstain in multicolor fluorescent techniques. Flow cytometry protocol for staining of dna with propidium iodide for cell cycle analysis using ethanol fixation. Pi is often used as a counterstain in multicolor fluorescence. Cells exhibiting fluorescence above 630 nm should be excluded from further analysis. Binding of propidium iodide to dna causes a red shift of the excitation maximum to 540 nm and the emission maximum to 640 nm, with a two to threefold increase in fluorescence.
Propidium iodide or pi is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Propidium iodide protocol for adherent cells for livedead cell imaging. Protocol aim the aim of this protocol is to provide instructions for performing live dead staining using calcein am and propidium iodide. When excited by 488nm laser light, it can be detected with in the petexas red channel with a bandpass filter 61010. Pi is membrane impermeant and generally excluded from viable cells. Perform all other staining as per your standardized method. The disadvantage is that it is often incompatible with fluorescent proteins and some surface markers. Fluorescence excitation and emission profiles of propidium iodide bound to dsdna. Common markers incompletely visualize the cell organization in the developing cns. Guidelines for use staining cells with propidium iodide for fluorescent. These dyes cannot pass through intact cell membranes. Bacterial viability kits now allow researchers to easily, reliably. Assessment of neuronal viability using fluorescein diacetate. Because pi is excluded from viable cells, it often used to selectively stain dead cells in a mixed livedead cell population.
Cell cycle analysis by dna content protocols flow cytometry at uc san diego moores cancer center. I would like to identifiy necrosis by fluorescence microscopy in my wound tissue samples mouse. The result is red fluorescence surrounding each cell in the root. Read how propidium iodide staining can be used to assess cell cycle state using flow cytometry. Dear sirmadam, i am enquiring about your propidium iodide ab14083. Propidium iodide nucleic acid stain life technologies pages. Dna staining for fluorescence and laser confocal microscopy. This kit is valuable for researchers looking to assess the. Annexin v staining assay protocol for apoptosis abcam. Procedure for the early detection of apoptosis using annexin v staining, and optional propidium iodide pi. The photomicrograph below illustrates the dnaintercalating probe propidium iodide in a nuclear preparation using the red longpass wg fluorescence cube. Viability staining with syto9 and propidium iodide pi is a frequently used tool in microbiological studies. Therefore, protocol 2 was developed, which uses sytox green to identify nonviable bacteria and dapi to detect the total bacterial population.
Fluorescence microscope cytometer or cell counter centrifuge slide glass, cover glass, or chamber slide reagents required materials cell staining reagents the following is a general protocol for preparing. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a. Pi cannot cross intact plasma membrane and therefore will only be present in dna of cells where the plasma membrane has been compromised permeabilized. Propidium iodide pi belongs to the same chemical class of ethidium bromide. Propidium iodide staining underestimates viability of. I am going to use propidium iodide to differentiate between live and dead cells using fluorescence microscopy after chemical compound treatment of adherent cells. 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 1,2.
In this application note we present a staining protocol using fluorescein diacetate fda and propidium iodide. Pi is commonly used for identifying dead cells in a population and as a counterstain in multicolor fluorescent techniques. Description propidium iodide pi is an ethidium bromide analog that emits red fluorescence upon intercalation with doublestranded dna. Cell membrane integrity excludes propidium iodide from staining viable and apoptotic cells.
Is this product suitable for using on fixed tissue samples. M propidium iodide merck h 2 o solution, with 0100. Please read the following cell viability protocol in its entirety before beginning. Propidium iodide pi and hoechst 33342 double staining openi. In this application note we present a staining protocol using fluorescein diacetate fda and propidium iodide pi, which stain viable cells and dead cells, respectively. Cellular viabilitycalcein propidium iodide johns hopkins. These stains differ both in their spectral characteristics and in. The calcein assay is based on the conversion of the cell permeant nonfluorescnt calcein am dye to the fluorescent calcein dye by intracellular esterase activity in live cells. Annexin vfitc apoptosis detection kit features annexin vfitc apoptosis detection kit contains readytouse solutions, annexin vfitc conjugate, propidium iodide pi. Caymans annexin v fitc assay kit employs a fitcconjugated annexin v as a probe for phosphatidylserine on the outer membrane of apoptotic cells. Propidium iodide pi is a cell impermeable nucleic acid intercalating dye.
What is the correct protocol for a livedead assay in a fluorescence based wellplate reader using calcein am and propidium iodide. Binding of propidium iodide to dna causes a red shift of the excitation maximum to 540 nm and the emission maximum to 640 nm, with a two to threefold increase in fluorescence intensity. The signals from the dyes are typically measured using fluorescence microscopy, fluorescence based microplate readers or flow cytometry. Suitable for use in fluorescence microscopy, confocal laser scanning microscopy. What is the correct protocol for a livedead assay in a. Pi staining is a crucial implement in fluorescence analysis of postnatal tissues. A number of fluorescent stains are available that label dna and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including hoechst, 4. Propidium iodide is a suspected carcinogen and should be handled with care. Introduction about this assay caymans annexin v fitc assay kit employs a fitcconjugated annexin v as a probe for phosphatidylserine on the outer membrane of apoptotic cells. When in an aqueous solution, pi has a fluorescent excitation maximum of 493 nm bluegreen, and an emission maximum of 636 nm red. We examined five nucleic acid binding fluorescent dyes, propidium iodide, sybr green i, yopro1, toto3, and topro3, for nuclear dna staining, visualized by fluorescence and laser confocal microscopy. Application note we present a staining protocol using fluorescein diacetate fda and propidium iodide pi, which stain viable cells and dead cells, respectively. Senckenbergische anatomie, institut fur anatomie ii, goetheuniversitat frankfurt am main, germany.
Staining procedure for a fluorescence microscopy for 24well plate. Cells emitting blue fluorescence were hoechst positive while those with red fluorescence were pi positive. Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometrybased methods. This protocol describes how to accurately measure neuronal viability using fluorescein diacetate fda and propidium iodide pi double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research. The methods used for detecting apoptosis were light microscopy may grunwaldgiemsa and trypan blue staining, fluorescence microscopy acridin orangeethidium bromide and annexin v propidium iodide. Propidium iodide pi and hoechst 33342 double staining in cultured c6 cells 72 hours after 400. Propidium iodide pi is a popular redfluorescent nuclear and chromosome counterstain. The fluorescence excitation maximum is redshifted for 3040 nm and the fluorescence emission maximum is blueshifted for 15 nm or so. Pi staining is a crucial implement in fluorescence.
Confocal microscopy is an excellent tool for imaging at cellular resolution. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. Propidium iodide excitation 536 nm, emission 617 nm can be used for routinary analysis of nuclear. Root tips of oryza sativa cv nipponbare rice seedlings 3, 6 and 8 days after germination were incubated for 10 min in a 10. Propidium iodide pi is a popular red fluorescent nuclear and chromosome counterstain. Propidium iodide nucleic acid stain 2 before you begin materials required but not provided see the protocols below to determine materials required for your particular use of pi. We counterstain with propidium iodide pi, a nucleic acid binding dye that is excluded from the membranes of healthy cells. Propidium iodide is suitable for fluorescence microscopy, confo cal laser scanning microscopy, flow cytometry and fluorometry. Pi is suitable for fluorescence microscopy, confocal laser scanning micro scopy, flow cytometry, and fluorometry. Inverted fluorescence microscope with filter sets for texas red and fitc. Fluorescence microscopy methods for determining the viability. Fluorescence microscopy 2x ssc dnasefree rnase antifade reagent, such as slowfade gold s36936 or prolong gold p36930 antifade reagents. Pi is commonly used for identifying dead cells in a population and as a counterstain in multicolor fluorescent. Nucleus staining fluorescent dyes with aromatic amino or guanidine groups, such as propidium iodide pi, ethidium bromide eb, diaminophenylindole dapi, acridine orange ao, and hoechst dyes, interact with nucleotides to emit fluorescence.
Fluorescence based assays are extensively used in microbial viability assessment, especially for mixedspecies samples or. Since its introduction, the propidium iodide pi flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. Concentration of propidium iodide for livedead staining of s. Protocol propidium iodide cell cycle staining protocol. Membrane impermeant, phenanthridinium intercalator dye excited by mercury or xenonarc lamps or argonion laser. Add this solution to 1 ml of a cell suspension containing 10,000,000 cells. Viability staining protocol calcein am and propidium iodide. Propidium iodide pi bluegreen lasers, also stains rna dapi uvviolet lasers.
The kit can identify apoptotic and necrotic cells detect by flow cytometry or fluorescence microscopy no need to fix cells mechanism. Cells will only be stained if the membrane has been. The kit can identify apoptotic and necrotic cells detect by flow cytometry or fluorescence microscopy. There is at most a twofold difference in fluorescence. Propidium iodide stock solution 50 gml ribonuclease i stock 100 gml method. The methods used for detecting apoptosis were light microscopy may grunwaldgiemsa and trypan blue staining, fluorescence microscopy acridin orangeethidium bromide and annexin v propidium iodide staining and agarose gel electrophoresis of fragmented genomic dna. The cyto 9 is being taken up and imaging really well but the. The appearance of phosphatidylserine ps residues normally hidden within. Concentration of propidium iodide for livedead staining. Suitable for use in fluorescence microscopy, confocal laser scanning microscopy, flow cytometry, and fluorometry. Pi can only cross compromised bacterial membranes and is. Propidium iodide pi is membrane impermeant and therefore does not enter viable cells with intact membranes. The staining protocol is applicable to adherent cells, single cells embedded in extracellular matrix and 3d cell clusters, for example multicellular spheroids. In the absence of cells, propidium iodide exhibits an excitation maximum near 500 nm and an emission maximum near 625 nm.
Analysis of apoptosis by propidium iodide staining and. Propidium iodide nucleic acid stain university of illinois. A protocol combining multiphoton microscopy and propidium. The aim of this study was to explore a novel cellular staining protocol using acridine orange propidium iodide aopi and to test the antiproferative activity of vad against human lung cancer a549 cells and human prostate cancer pc3 cells. Apr 24, 2019 propidium iodide pi is widely used for bacterial viability staining, especially since boulos et al. Propidium iodide staining of cells for cell cycle analysis. Simultaneous use of two fluorescent dyes allows a twocolor discrimination of the population of living cells from the deadcell population. Propidium iodide pi is membrane impermeant and generally excluded from viable cells. A common alternative method of analysis involves using fluorescent dyes syto 9 and propidium iodide pi which differentially stain live and dead bacteria. Pi staining led to similar staining patterns as hemalaun in bright field microscopy. Protocol for counterstaining cells with propidium iodide. The samples were further studied by fluorescence microscopy for quantitative and qualitative analysis.
However, this protocol cannot be combined with immunofluorescence for host proteins, due to the fluorescence of propidium iodide in two of the four channels available for conventional fluorescence microscopy. Pi can be excited with a xenon or mercuryarc lamp or with the 488 line of an argonion laser. The optimal concentration, costaining of rna, and bleaching speeds were examined. Pdf files, either single images or for the final assembled figure for a paper. Immediately before flow cytometric analysis, prepare a 0. Critical aspects of using bacterial cell viability assays. It is commonly used in evaluation of cell viability or dna content in cell cycle analysis by flow cytometry. Propidium iodide is suitable for fluorescence microscopy, confocal laser scanning microscopy, flow cytometry and fluorometry. Experimental protocolscounterstaining adherent cells for fluorescence microscopy preparing the sample use the fixation protocol appropriate for your sample. Viability staining protocol calcein am and propidium iodide this is a suggested procedure, please adjust according to your experimental needs.
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