Journal of Microscopy, 2014

doi: 10.1111/jmi.12133

Received 7 January 2014; accepted 9 April 2014

Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques N . A T A L E ∗, S . G U P T A ∗, U . C . S . Y A D A V † & V . R A N I ∗

∗ Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Uttar Pradesh, India

†Department of Biochemistry & Molecular Biology, UTMB Galveston, Texas, U.S.A.

Key words. Apoptosis, necrosis, nuclear aberrations, staining techniques.

Summary Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst-33342, 4’, 6-diamidino-2-phenylindole (DAPI), Acridine orange–Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin-V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells.

Introduction Apoptotic cell death is a highly regulated process that is characterized by distinct cellular morphology and various biochemical changes. It includes chromatin condensation, nuclear fragmentation, reduction in cell volume and formation of surface vesicles or membrane blebbing. At the Authors Atale and Gupta have contributed equally. Correspondence to: Vibha Rani, Department of Biotechnology, Jaypee Institute of Information Technology, A-10 Sector-62, Noida, 201307, Uttar Pradesh, India. Tel: + 91-120-2594207 fax: +91-120-2400986; e-mail: [email protected]

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molecular level, extensive DNA strand break, degradation, cross-linking and relocation of proteins and DNA fragments take place (Frey, 1995). Although apoptosis is important in maintaining, homeostasis of the body, development of organs and removing the damaged cell, exogenously induced and excessive apoptosis, or lack of it, is linked with many diseases such as degenerative disorders (Fink & Cookson 2005; Favaloro et al., 2012). Therefore, techniques to precisely identify the nature of cell death are important in understanding the basic mechanisms underlying the pathophysiology of the process. Apoptosis, induced by several stress and cell-death signals, can take place by two main pathways, the extrinsic or death receptor (tumor necrosis factor-induced/Fas–Fas ligand–mediated) and the intrinsic or mitochondrial (small mitochondria-derived activator of caspases-induced) pathways, yet the molecules in one pathway can influence the other (Jeon, 2002). The initiator caspase molecules are cleaved proteolytically in series in which one caspase activates the other, eventually leading to DNA fragmentation, degradation of cytoskeletal and nuclear proteins and formation of apoptotic bodies (Elmore, 2007). The cells also die by other methods such as necrosis, which is caused by sudden abnormal change in the cell’s environment, including extremely harsh external factors such as physical or chemical damage to the cells or tissue. Unlike apoptosis, necrosis cells die and release their content, which include highly harmful lysosomal proteolytic enzymes that affect other cells and start a cascade of cell-death event, in the surroundings. Cell death by necrosis is known to result in gangrene. Similarly, cell death by viral infection and phagocytosis has its own characteristics (Alberts et al., 2002). In phagocytosis, the dying cells are phagocytosed by the immune cells; however, in many autoimmune diseases, even healthy cells are phagocytosed and killed, for example, in type-1 diabetes, islets cells are killed by phagocytosis. DNA plays a unique role in the cellular and nuclear biology, affecting key regulatory steps in the healthy balance and proper functioning of the cell. However, many extra- and intracellular events change the integrity of DNA by either directly damaging it or indirectly modifying its structure and function

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Fig. 1. Schematic presentation of different staining techniques.

(Surova & Zhivotovsky, 2013). These changes invariably lead to an effect first on the nucleus and then on the cell, which is sometimes detrimental to the cells resulting in cell death via apoptosis or necrosis. To precisely diagnose and differentiate different types of celldeath methods, very sensitive techniques have been developed. These diagnostic tools aim at identifying the characteristic molecules associated with different types of cell death, thus clearly marking the method of cell death, which likely indicates the cause of the cell death. For example, in apoptosis an early and critical event involves inside-out inversion of cell membrane exposing phosphatidylserine onto the surface, which helps in recognition and uptake of apoptotic cells by phagocytes. These early events can be identified by Annexin-V staining, which bind specifically to phosphatidylserine molecules. In some cases, the changes on the surface of apoptotic cells are difficult to detect, but stains or dye that can directly bind to DNA can depict the method of death in a specific manner (Zhang et al., 1997). Several other characteristic of apoptotic cells, such as loss of cell viability, DNA fragmentation and DNA condensation, are useful traits to monitor apoptosis (Ziegler & Groscurth, 2004). Similarly, there are markers of other methods of cell death that serve as target molecules in diagnostic tools. In this review, we describe a range of cytosolic or nuclear vital fluorescent and nonfluorescent staining techniques, which help to understand the working methodology and precautions that can be utilized to assay the cellular and nuclear markers, and to precisely detect the cause of cell death (Fig. 1).

Fluorescent nuclear stains to analyse cell death 4’,6-Diamidino-2-phenylindole (DAPI) staining. DAPI is a blue fluorescent dye that preferentially stains dsDNA by strongly binding to adenine-thymine–rich regions. Binding of this stain produces an enhanced fluorescence due to the displacement of water molecules from both DAPI and the minor groove of the DNA. However, in a different binding mode, which involves adenine-uracil dinucleotide-selective intercalation, it also binds RNA. The DAPI/RNA complex exhibits a longer wavelength fluorescence emission, more than the DAPI/dsDNA complex (Hoff, 1988; Gomes et al., 2013). When bound to DNA, it has absorption maxima at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore for fluorescence microscopy, DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. DAPI is used mostly in fluorescence immune staining as a counter stain. After the immunostaining steps are completed, the sample is briefly washed with phosphate buffered saline solution (PBS). Working stock (50 μg mL−1 ) is prepared by appropriately diluting the DAPI stock solution in PBS. The cells are incubated in the stain for 2–5 min. Samples are rinsed two to three times with PBS and excess buffer is subsequently drained out. Samples are mounted with the cover slip and slides are examined under the fluorescence microscope with appropriate filters. DAPI is a nuclear counter stain used in multicolour fluorescent techniques for cell culture samples to detect DNA of contaminating mycoplasma, virus and cell death mechanism

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(apoptosis). Its blue fluorescence stands out in vivid contrast to green, yellow or red fluorescent probes of other structures (Barcellona & Gratton, 1996). Nuclear changes such as nuclear fragmented bodies, condensed or deformed nuclei can be visualized by DAPI during apoptosis or necrosis (Atale et al., 2013). The fluorescence intensity of eluted stain can also be measured during various experimental conditions. Further, flow cytometric evaluation of cellular homogenates validates high or low amount of DNA content compared to control cells. Its selectivity for DNA and high cell permeability allows efficient staining of nuclei with little background from the cytoplasm. Based in this property, DAPI can be added in the mounting medium itself and additional staining steps can be ruled out. It is a classic nuclear dye for immuno-fluorescence microscopy as well as an important component of high-content screening methods requiring cell-based DNA quantitation. DAPI can be used for both fixed and live cell staining, though the concentration of the stain needed for live cell staining is generally much higher than for fixed cells. As it is a DNA binding compound it is likely to have some low-level mutagenic properties and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations. Propidium iodide staining (PI). PI [P-1304 (solid); P-3566 (solution)] is a red-fluorescence dye with excitation/emission maxima 535/617 nm with bound DNA and is permeant only to dead cells (Nicoletti et al., 1991). PI is impermeable to cells with an intact plasma membrane (PM), hence when the cell integrity becomes compromised it gains access to the nucleus where it complexes with DNA rendering the nucleus highly fluorescent (Brana et al., 2002). PI binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per four to five base pairs of DNA. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining (Nockera et al., 2006). PI staining is performed varyingly depending upon the downstream usage. Adherent cells are stained with other fluorescent methods followed by staining with diluted PI (1.5 mM) solution. Samples are then mounted in a medium with an antifade agent and observed under fluorescent microscope (Riccardi & Nicoletti, 2006). For suspension cells, PI staining is used for fluorescence activated cell sorter analysis by flow cytometry. The cells with or without other staining, such as monoor multicolour fluorescence stain for cell receptors, are suspended in PBS and centrifuged at room temperature. Direct DNA staining in PI hypotonic solution is the quick method for thymocytes and nonadherent mononuclear cells. Further, PI staining after alcoholic fixation is the standard method for adherent cells and multinuclear cells growing in suspension. Cells are then analysed by flow cytometry (Krishan, 1975).  C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy 

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For providing distinction between dead and apoptotic cells, as membrane impermeable DNA stain, PI is added simultaneously to the cell suspension (Lopez-Amoros et al., 1995). PI is suitable for fluorescence microscopy, immunohistochemical characterization of labelled cells, confocal laser scanning microscopy (Laake et al., 1999), flow cytometry (Troiano et al., 1998) chromosome FISH (Rab et al., 1996) and fluoresceinlabelled antibodies allowing simultaneous detection of nuclear DNA. PI is toxic, thus, gloves, protective clothing and eyewear should be worn and safe laboratory practices must be followed. PI is a known mutagen (Edwards et al., 2007). Solutions containing PI should be poured through activated charcoal before disposal. The charcoal must then be incinerated to destroy the dye (Ashworth et al., 1994). Hoechst 33342 staining. Hoechst 33342 (2’-[4ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5’-bi-1Hbenzimidazole trihydrochloride trihydrate) is a cell-permeable DNA stain that is excited by ultraviolet light and emits blue fluorescence at 460–490 nm. It binds preferentially to adenine-thymine regions of DNA. This stain binds into the minor groove of DNA and exhibits distinct fluorescence emission spectra that are dependent on dye:base pair ratios. Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA (Latt et al., 1975; Sibirtsev et al., 1997). There are three related Hoechst stains: Hoechst 33258, Hoechst 33342 and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra. Hoechst 33342 can be used to quantitate DNA in solution. The method is relatively insensitive at pH 5–10 but is sensitive to temperature and ionic strength changes and fluorescence quenching by divalent or heavy metal cations (Regina, 2006). The cells are centrifuged and resuspend in buffered salt solutions or media, with optimal dye binding at pH 7.4. The adherent cells can be stained in situ on cover slips. When performing for the first time, it is advisable to try several dye concentrations over a range (0.2–5 μg mL−1 ) to determine the concentration that yields optimal staining. Add the Hoechst stain to the cells using the best concentrations determined. Incubate for 10–30 min. Wash the cells with PBS and examine under the fluorescence microscope or by flow cytometry. Hoechst 33342 is used for specifically staining the nuclei of living or fixed cells and tissues. This stain is commonly used in combination with 5-bromo-2’-deoxyuridine (BrdU) labelling to distinguish the compact chromatin of apoptotic nuclei, to identify replicating cells and to sort cells based on their DNA content. A combination of Hoechst 33342 and PI have been extensively used for simultaneous flow cytometric and fluorescence imaging analysis of the stages of apoptosis and cell-cycle distribution. This stain is also often used as substitutes for another nucleic acid stain called DAPI.

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Hoechst dyes are commonly used to stain genomic DNA in applications such as fluorescence microscopy and immunohistochemistry, often together with other fluorophores (Johnson & Michelle, 2011), flow cytometry to count or sort out cells, detection of DNA in the presence of RNA in agarose gels (Mocharla et al., 1987), automated DNA determination and chromosome sorting (Sterzel et al., 1985). Hoechst stains bind to DNA, hence it sometimes interferes with DNA replication during cell division. Consequently, they are potentially mutagenic and carcinogenic, so care should be taken in their handling and disposal. Hoechst 33342 and DAPI or PI double staining. Hoechst 33342 is a blue-fluorescence DNA-specific dye that stains the condensed chromatin in apoptotic cells (Schmid et al., 2007). Early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared to live cells because of changes in membrane permeability (Schmid et al., 1992). Hoechst 33342 can also be used in double stain apoptosis detection kit with PI (Darzynkiewicz & Traganos, 1997). PI is a red-fluorescence dye that is only permeant to dead cells (Berney et al., 2007). The staining pattern resulting from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic and dead cell populations by flow cytometry and fluorescence microscopy. The single cell suspension cells are centrifuged and resuspended in 1 mL prewarmed (37°C) PBS–bovine serum albumin. HO342 stock solution is added and the mixture is gently vortexed. Cell suspension is then placed on ice, and PI stock solution is added and incubated again. The samples are run on the flow cytometer. HO342 and 7-AAD fluorescence is collected in log mode (Olive et al., 1992; Allen et al., 2001; Foglieni et al., 2001). Live cells are represented as red dots, early apoptotic cells show up as green dots, late apoptotic cells are represented in blue and cells with degraded DNA appear violet when observed under flow cytometer (Mpoke & Wolfe, 1997). Hoechst 33342 and DAPI double staining provide more reliable quantification of dying cells, not requiring sample fixation or special buffers and reagents (Monger & Landry, 1993). Necrotic cells can be discriminated from cells undergoing apoptosis, making the HO342/7-AAD method applicable to experimental systems where both modes of cell death may occur simultaneously (Bottiroli et al., 1989). It is used for staining neuronal nuclei in apoptosis induced by CGA-activated microglia (Ciesielski-Treska et al., 2001). Higher concentrations of HO342 staining dye may alter dye binding and create a red shift of the emission spectrum. The incubation period at 37°C must not be exceeded because discrimination between live and apoptotic cells becomes less distinct. Adding higher concentrations of 7-AAD will increase background staining (Pomar et al., 2005; Petriz, 2007). Acridine orange (AO) – ethidium bromide (EB). AO and EB are intercalating, nucleic acid-specific fluorochromes, which emit

a green and orange fluorescence, respectively, when bound to DNA (Petit et al., 1993). Of the two, only AO can cross the PM of viable and early apoptotic cells. EB is only taken up by cells when cytoplasmic membrane integrity is lost (Kasibhatla et al., 2006). An AO/EB-based assay combines the conventional, and for apoptotic quantification minimizes adherent cell damage, decreasing possibility of floating cells and reducing the time of detection (Ribble et al. 2005). In fluorescence microscopy, viable cells appear to have a green nucleus with intact structure whereas apoptotic cells exhibit a bright green nucleus showing condensation of chromatin as dense green areas. Thus live cells have a normal green nucleus; early apoptotic cells have bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; cells that have died from direct necrosis have a structurally normal orange nucleus (Bishell & Shiigi, 1980). Suspension cell cultures and adherent cell’s supernatant (medium and floating cells) and the detached adherent cells (with PBS–ethylene diamine tetraacetic acid, Dulbecco’s PBS) from the same sample are pooled together. The cells are pelleted by centrifugation and washed with cold PBS once. Cell pellets are then resuspended in 25 μL cold PBS, and 2 μL EB/AO dye mix is added. Stained cell suspension (10 μL) is placed on a clean microscope slide and covered with a cover slip and can be visualized (Schmued et al., 1982). The conventional EB/AO method can be modified by centrifuging the cells in a 96-well plate to bring down all the cells, including floaters, to the bottom of the plate. EB/AO staining assay method substitutes the detaching and washing steps with a simpler centrifugation step with 96-well plates. This technique is easy to perform, time efficient (5–10 min) and suitable for adherent cells. AO staining of unfixed cells may be used as a simple, fast means of obtaining information on cell ploidy levels, cell cycle status from DNA measurements (green fluorescence), cell transcriptional activity from RNA staining (red fluorescence), human and murine cell lines, peripheral blood and bone marrow specimens from patients with leukemia and mitogenically (phyto-hemagglutinin) or antigenically (mixed lymphocyte culture) stimulated human peripheral blood cultures (Ferlini et al., 1996), staining apoptotic and necrotic human neurons in bacterial meningitis (Braun et al., 1999) assessment of apoptosis and viability of polymorphonuclear neutrophil (PMNL) by fluorescent microscopy after staining with AO/EB (Pitrak et al., 1996) and staining of GDC-induced apoptotic hepatic cells (Kwo et al., 1995). After AO/EB staining, viable cells appear green with intact nuclei, early apoptotic cells stain greenish orange and late apoptotic cells appear reddish orange with condensed and fragmented nuclei. The variability of nuclear fluorescence indicates the anomalies and morphological alterations than healthy cells. Any accidental exposure to EB may be fatal if inhaled, harmful if swallowed or absorbed through skin, causing  C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy 

FLUORESCENT AND NONFLUORESCENT DNA STAINING TECHNIQUES

irritation to skin, eyes and the respiratory tract and causing heritable damage. AO may also cause discomfort if swallowed, prolonged skin contact may cause redness and eye irritation. ssDNA staining. ssDNA immunostaining differentiates between necrotic and apoptotic cell death by visualizing ssDNA fragments in the nuclei using a murine monoclonal antibody (IgM - mAb 3299) to ssDNA. This antibody does not recognize DNA in double stranded conformations, and provides early specific detection of apoptosis (Orlowski & Dees, 2003). Apoptosis-specific ssDNA apostain method is also used for staining ssDNA (Kaldawy et al., 2002). Fluorescence for the cyanine stilbene dyes (Pico Green and SYBR Green I) allow differentiation between ssDNA and dsDNA by measuring ssDNA/dsDNA ratios (Cosa et al., 2001; Elmendorff-Dreikorn et al., 1999). Pico Green’s preferential complexation with DNA is explained by binding a constant for a dye with ssDNA, as it is lower than that with dsDNA (Arndt-Jovin & Jovin, 1989). Even at a 1:10 dye–DNA ratio, the fluorescence contribution is negligible (Zubay et al., 1995). However, increasing DNA concentration to 100-fold increases the fluorescence. Further at higher ssDNA content renaturation would occur faster, producing more dsDNA sites and expanding concentration dependence (Cosa et al., 2000). Pico Green is well suited for the analysis of DNA damage since its decay is monoexponential in the presence of dsDNA. Lifetime for dsDNA is much longer than that for ssDNA and it intercalates into both ss-DNA and dsDNA. The effects of high dye concentrations indicate that the dimers observed in CD spectra are responsible for the energy transfer or self-quenching decay observed in fluorescence time-resolved studies (Rogers et al., 1999). The formation of dimmers is higher in ssDNA than dsDNA and therefore dye–ssDNA complexes are more prone to undergo energy transfer deactivation than complexes formed with dsDNA, thus explaining the success of DNA damage assessment techniques. Pico Green when complexed with DNA induces circular dichroism (ICD). The CD spectrum for dye–dsDNA and dye– ssDNA (1:10) shows a negative band, which coincides with the absorption of Pico Green with intensity for dye–ssDNA, which is 2/3 of dye–dsDNA. Results obtained under high dye–DNA ratios (i.e. 1:1) showed that a negative peak is slightly red shifted and the band is much narrower than that at low dye–DNA ratios in dsDNA. For ssDNA, the resulting CD spectrum at high dye–DNA ratio showed a 2/1 pattern with increasing wavelength, changing sign at 506 nm (Kubista et al., 1987; Baumstark-Khan et al., 2000). In ssDNA immunostaining, cells are stained at room temperature and given PBS washes after incubation (Cantor & Schimmel, 1980). Cover slips are incubated in methanol:PBS (6:1) for 2 min on ice, boiled in 1.25 mM MgCl2 for 5 min, then placed back on ice for 10 min. Nonspecific staining is blocked by incubation in 10% FBS in PBS for 30 min on ice. Primary antibody (mouse IgM mAb/anti-rabbit Ig /F 7–26/ Ki  C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy 

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67 to ssDNA) is diluted to a final concentration of 10 mg mL−1 in FBS–PBS, and cover slips are incubated for 30 min on ice followed by 30 min at room temperature (Frankfurt et al., 1997; Sugawara et al., 2002; Zou et al., 2002; Nagata et al., 2004; Walters & Stegemann, 2014. Secondary antibody was donkey anti-mouse IgM-Cy-3 conjugated, diluted to 1:250 and applied for 20 min. Nuclei are visualized by staining for 1 min with Hoechst dye 33322 at a final concentration of 0.3 mg mL−1 in dH2 O, followed by fixation in 2% paraformaldehyde for 10 min. Cover slips are mounted on slides and examined on a Nikon Eclipse E600 (Nikon, Japan) fluorescent microscope (Akishima et al., 2005). In apostatin method, tissues on slides are boiled for 5 min in a 5 mmol/L MgC12 /PBS solution and placed on ice for 10 min. Nonspecific sites are blocked with 10% calf serum in PBS for 15 min on ice. Primary monoclonal antibody against ssDNA is incubated for 30 min at room temperature. Slides are rinsed twice for 5 min in PBS, followed by Vectastain Elite ABC reagent for 20 min. Slides are rinsed again for 5 min in PBS, then for 10 min in 0.05 mol/L Tris. Diaminobenzidine colour development was allowed to proceed for 5 min, the tissues were counterstained with methyl green and then mounted with cover slips (Studzinski & Benjamins, 2001). A sensitive and rapid in situ method is used to visualize sites on ssDNA in cultured cells and in experimental test animals. Anti-bromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the ss form. After treatment of cells with DNA-damaging agents or γ irradiation, ssDNA molecules form nuclear foci in a dosedependent manner within 60 min. The mammalian recombination protein Rad51 and the replication protein A then accumulate at sites of ssDNA and form foci, suggesting that these are sites of recombination DNA repair (Frankfurt et al., 1996). ssDNA staining is used to assess the DNA damage for necrosis and apoptosis. 1X DNA denaturing buffer is toxic and irritant, therefore caution must be used. Protective gloves and clothing must be worn. SYTO probe staining. SYTO superfamily consists of four subfamilies spanning a broad range of visible excitation and emission spectra: (i) SYTO blue (Ex/Em 419–452/445–484 nm), probes SYTO 40–45; (ii) SYTO green (Ex/Em 483–521/500– 556 nm) probes SYTO 11–16 and 20–25; (iii) SYTO orange (Ex/Em 528–567/544–583 nm) probes SYTO 80–8, and (iv) SYTO red (Ex/Em 598–654/ 620–680 nm probes (Raderschall et al.,1999). Green fluorescent SYTO probes have been widely utilized in cytometric assays (Wlodkowic et al., 2008a; Wlodkowic et al., 2009). Cyanine SYTO probes allow noninvasive tracking of intracellular events with high-throughput analysis and livecell sorting for dynamic real-time analysis of apoptosis and

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detection of cell death using automated micro fluidic chipbased cytometry (Wlodkowic & Skommer, 2007). Fluorescence lifetime imaging with two-photon excitation of SYTO13 allows differential and simultaneous imaging of DNA and RNA in living cells (Eray et al., 2001). Straightforward staining and adaptability for automated dispensing, prolonged intracellular retention, lack of side-effects on cellular viability, proliferation or cell migration and lack of interference with the assay readout make SYTO-based assays a dynamic and high-throughput analysis technique (Zandvoort et al., 2002). SYTO reagents are diluted in DMSO and then stored in −20°C in dark. Cells were collected, rinsed with PBS and resuspended in 100 μL PBS containing selected SYTO dyes and PM permeability marker, PI (Sigma, 5 mg mL−1 ). After 20 min incubation at room temperature in the dark, 500 μL of PBS containing 2% FBS is added and cells are subjected to the flow cytometric analysis (Skommer et al., 2006a; Wlodkowic et al., 2007; Wlodkowic et al., 2011). Microfluidic chip–based cytometry system utilizes disposable chips run on bioanalyser (Skommer et al., 2006b; Wlodkowic & Darzynkiewicz, 2011). For on-chip cytometry, cells are stained with SYTO 16, centrifuged and resuspended in 100 μL of isobuoyant buffer; 10 μL cells are loaded to each sample. On-chip staining protocol may also be employed with SYTO and SYTOX probes diluted in isobuoyant buffer and added directly to chip wells loaded with cells. Two photon excitation inverted microscope system is equipped with a mode-locked titanium:sapphire (Ti:Sa) laser that produces 80-fs pulses at a repetition rate of 82 MHz (Bewersdorf et al., 1998; Wlodkowic et al., 2010). All fluorescence light below 700 nm is transmitted through the dichroic mirror and collected by a photomultiplier in photon counting mode. Additional blocking of excitation light is achieved by means of a series of 750-nm interference short pass filters in the imaging path behind the mirror. The method used to determine the fluorescence lifetime is based on time-gated detection of the fluorescence. After each excitation pulse, the fluorescence decay is detected in four time windows (gates) that are enabled sequentially, making the acquired lifetime intrinsically independent of laser intensity fluctuations. The signal from many excitation pulses is integrated for each pixel. The lifetime for every pixel results in the corresponding image (Kwak et al., 2001; Nielsen et al., 2001; Broess et al., 2009). SYTO probes overcome low cell number obstacle by the dynamic analysis of samples, for example, primary cancer stems cells. Cyanine SYTO probes rapidly diffuse through eukaryotic membranes and are applicable to many polychromatic assays in studies of caspase-dependent cell death (Zhao et al., 2012). SYTO probes are also used for mammalian cells nuclear morphology visualization (SYTO 13) and assessment of DNA replication (SYTO 24) after exposure to natural and synthetics estrogens (Andreescu et al., 2005; Wlodkowic et al., 2008b). SYTO probes can be effective substrates of multi drug resis-

tance efflux pumps (e.g. P-glycoprotein; Holmstrom et al., 1998; Van der Pol et al., 2003). No data are available addressing the mutagenicity or toxicity of these reagents. Because the reagents bind to nucleic acids, they should be treated as potential mutagens and used with appropriate care (Westers et al., 2005).

Nonfluorescent stains Methyl green staining Methyl green dye is a nuclear counter stain designed to be used after completion of immunohistochemical staining. This dye is specific for chromatin and has a strong affinity for highly polymerized DNA (Haugland, 2005). The action of DNase on this complex results in the loss of this affinity for methyl green. Furthermore, the visible colour of the complex is stable at pH 7.5 whereas the colour of free methyl green fades to colourless in about 12 h. These characteristics form the basis for the colorimetric assay of DNase by measuring the rate of disappearance of the colour of the DNA–methyl green complex. It stains DNA green to blue (Lyon et al., 1989). Slides are prepared and then rinsed with tap water followed by staining with methyl green counter stain. Stained slides are subjected to an incubation temperature of 60°C for 1–5 min. After incubation, slides are rinsed with deionized water until the rinsed water is clear. Excess water is removed by tapping. Slides are dipped in acetone containing 0.05% acetic acid 5–10 times and then immediately dehydrated through 100% ethanol. Slides are mounted and observed under a light microscope. The nuclear chromatin in the nucleus is composed of nucleoproteins, that is, DNA, found in the nucleus and RNA in the nucleolus and cytoplasm. DNA functions in cell heredity, and synthesis of RNA. RNA functions principally in protein synthesis so this stain is used for staining special cells and tissues, specifically frozen dried tissues (Kunitz, 2009). General precautions should be followed for safety, like wearing of lab coats and gloves while working with this stain. Any kind of contact and inhalation of the dye should be avoided (Mohtasham et al., 2010).

Haematoxylin–eosin staining (H&E) Haematoxylin (basic) and eosin (anionic) are widely used in histopathology where the nucleus is stained with hematoxylin and cytoplasm by eosin (Moore et al., 2003; Fischer et al., 2008; Sampieri et al., 2013). A basic (cationic) solution is used to stain nuclei [DNA, RNA and acid nucleoprotein (rich in arginine), followed by an acid (anionic) solution that stains cytoplasm (basic protein; Martin et al., 1987)]. The dyes have different affinities for different sites in the cell and bind to them electro statically, resulting in salt formation. There is no  C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy 

FLUORESCENT AND NONFLUORESCENT DNA STAINING TECHNIQUES

chemical interaction between nuclear and cytoplasmic staining solutions. Fixation of cells is required for staining and cytological diagnosis. The factors in fixation that influence cell staining are cell preservation, stabilization, prevention of cell contents loss, revealing of reactive sites for staining and permeabilizing the cell membrane for the dyes (Drachenberg et al., 1997). Fixed samples are stained by H&E dye. The nucleolus is stained red when wet-fixed and rehydrated smears were fixed in formol– alcohol (J¨orundsson et al., 1999). H&E staining can be performed using a modified rapid protocol for Laser capture microdissection. Slides are defrosted, fixed (70% ethanol for 1 min), H&E stained (Mayer’s hematoxylin for 30 s, Scott’s tap water for 10 s, eosin for 10 s) and dehydrated (70% ethanol for 30 s, 100% ethanol for 1 min, xylene for 2–5 min; Mayta et al., 2000). H&E solutions contained complete protease inhibitor cocktail. For processing experiments investigating the effect of different stages of the staining protocol on the protein profile, individual steps were omitted and replaced with mock incubations in water (Wang & Mushinski, 2006). H&E is the most commonly used general histochemical stain, for example, cervix or kidney cortex processing (Craven et al., 2002; Fenoglio et al., 2002). The lipotropic hormone triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, etc. (Liao et al., 1997). Haematoxylin has a blue-purple colour and stains nucleic acids. Eosin is pink and stains proteins nonspecifically. In a tissue, nuclei and nucleoli are stained blue, whereas the extracellular matrix and cytoplasm are stained pink. Solvents used in the staining are light-sensitive, can stain skin and clothing permanently and the stain causes health hazards. The dyes must be disposed in waste containers in fume hood. Trypan blue staining Trypan blue, a diazo dye is a vital stain used to selectively colour dead tissues or cells blue. Trypan blue is named so because it can kill trypanosomes, the parasites that cause sleeping sickness. Trypan blue is also known as diamine blue and Niagara blue and is derived from toluene. The reactivity of trypan blue is based on the fact that the chromophore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells that exclude the dye are viable. The dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell mem C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy 

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branes that exclude the dye, whereas dead cells do not. Since live cells are excluded from staining, this staining method is also described as a dye exclusion method. In this test, a cell suspension is simply mixed with dye and then visually examined to determine whether cells take up or exclude dye. A viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm (Koester et al., 1998). Live cells or tissues with intact cell membranes are not coloured. Hence, dead cells are shown as a distinctive blue colour under a microscope. Cells have to be diluted in complete medium without serum to an approximate concentration of 1 × 10 to 2 × 10 cells per mL, and 0.5 mL of a suitable cell suspension is taken in a test tube; 0.1 mL of 0.4% Trypan blue stain is added and followed by thorough mixing. The mixture is allowed to stand for 5 min at 15–30°C. Cell counting is done under hemocytometer and viable and nonviable cells are distinguished based on their colour (Strober, 2001). Trypan blue is commonly used in microscopy (for cell counting) and in laboratory mice for assessment of tissue viability. The method cannot distinguish between necrotic and apoptotic cells. It may also be used to observe fungal hyphae and Stramen. The stain is not thought to produce any adverse effects on skin or eye, or irritation of respiratory tract. Although, there is ample evidence that it can be regarded as being able to cause cancer in humans based on experiments and other information, and can also cause developmental toxicity. Cytosolic/extranuclear staining Nile blue sulphate staining Nile blue dye was found by a scientist Lorrain Smith in 1908 for distinguishing neutral fats, that is, triglyceride from fatty acids by staining fatty acids dark blue and triglycerides, steroids reddish pink. Nile blue is a combination of oxazine sulphate (true nile blue) and the oxazone (nile red; Goto, 1987). Nile blue A (Nile blue sulphate) is a basic oxazine dye, which is soluble in water and ethyl alcohol (Lin et al., 1991). The oxazone form of the dye (Nile pink) is formed by the spontaneous oxidation of Nile blue A in aqueous solution or by refluxing Nile blue A with dilute sulphuric acid. Nile pink is soluble in neutral lipids, which are liquid at the staining temperature (Lyon, 2000). The Nile blue A–Nile pink mixture has been used as a histological fat stain in prepared tissue sections (Wick, 2012). In polar media, its absorption and emission maxima shift to red, which is indicative of stabilized charge separation in the excited state. Consequently, this dye has been used to monitor events that depend upon solvent polarity. Nile blue tends to have a higher affinity for cancerous cells than healthy ones and it is a photosensitizer for oxygen – these two properties can be useful for photodynamic therapy (Ostle et al., 1982). The tissues and cells that are examined by this stain consist of adipose tissue and adrenal cortex of the rat, sebaceous glands,

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fat cells and stomach epithelium of the mouse. Sections are cut and stained in 1% aqueous solution of Nile blue for 5 min, differentiated in 1% acetic acid for 30 s, washed and mounted. In certain cases 0.2% solution of Nile blue can be used. It can also be used for staining colonies on agar plate. Staining solution of Nile blue sulphate is prepared by dissolving 0.05 g of Nile blue sulphate in 100 mL of ethanol. Colonies on the agar plate are stained with 5 mL of staining solution and shaken gently at room temperature. After 20 min, staining solution is removed from the agar plate and the plate stood to dry the surface (Jose et al., 2009). Nile blue and Nile red dyes are fluorescent stains used to stain normal and pathological skeletal muscle fibres. In normal muscle, lipid storage disorders and mitochondrial myopathies, Nile blue stains the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red–stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibres and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies (Kitamura & Doi, 1994). The stain is also used to detect apoptosis where cells have faint blue background stained areas as compared to the dark blue staining in regions of cell death. The most useful spectroscopic parameter of the dyes is their fluorescence at relatively long wavelengths, 680 nm, in aqueous media. Probes that emit above 650 nm are relatively few, yet they tend to be the most useful ones for tissue and intracellular imaging applications (Bonilla & Prelle, 1987; Changxia et al., 2004). There are insufficient data available on the toxic effects of this dye. However, other dyes in this group are known to be potential carcinogens, mutagens and highly toxic. Hence, this dye has to be handled with care as it is a skin, eye and respiratory tract irritant. The affected area on the skin has to be washed with soap or mild detergent and large amounts of water until all evidence of the chemical has been removed (approximately 15 min). If irritation persists seek medical attention, wash contaminated clothing before re-use. If inhaled accidentally, then applying artificial respiration is another option to provide relief to the patient.

Annexin-V – fluorescein isothiocyanate (FITC) conjugated staining Biotin or FITC labelled Annexin V detects apoptosis by targeting the loss of phospholipid asymmetry in PM in tissues, embryo or cultured cells in the presence of Ca2+ ions (Frangioni, 2003). Since, loss of PM asymmetry is an early event in apoptosis, Annexin V interacts strongly and specifically with the exposed phosphatidyl serine (PS) residues

of the outer PM leaflet by measuring PS exposition in tissues, embryos or cultured cells (Engeland et al., 1998). Extrinsically applied hapten (i.e. FITC or biotin)-labelled Annexin V, with absorption maxima of 492 nm and emission maxima 520 nm, detects apoptosis by binding in the presence of millimolar Ca2+ to PS residues that are exposed at the outer leaflet of the PM of apoptotic cells. Annexin V is not able to bind to normal vital cells since the molecule is not able to penetrate the phospholipid’s bilayer (Martin & Lenardo, 2001; Balaji et al., 2013). When examined by light microscopy, the cells showing morphological characteristics for apoptotic cells, for example, cellular shrinking, condensation and margination of the chromatin and ruffling of the PM, that is, ‘bud formation’ also show affinity for Annexin V (Koopman et al., 1994). Most of the cells, devoid of PI staining, indicate intactness of the membrane. When the membrane loses its integrity, the cell becomes both Annexin V and PI positive, indicating the necrotic stage of the cell (Vermes et al., 1995). For the quantification of Annexin V-positive apoptotic cells, flow cytometry can best be applied using a single cell suspension prepared from the cells or tissue under examination. Vital cells are negative for both PI and Annexin V; apoptotic cells are PI negative and Annexin V positive, whereas dead cells are positive for both PI and Annexin V. The Annexin V-labelled cells fixed in cold methanol enable detection and quantification of intracellular antigens. After fixation Annexin V binding is visualized with FITC-labelled streptavidin (Wetzel & Green, 2000). For in vivo or in situ detection of apoptotic cells, biotinlabelled Annexin V is injected in the bloodstream of animals followed by dissection, routine formalin fixation and paraffin embedding of the tissues. After dewaxing and rehydration, the tissue sections are incubated with peroxidase-conjugated strepavidin and visualized by enzyme histochemistry. Control sections incubated with Annexin V after fixation and embedding of the tissue are stained using peroxidase-conjugated streptavidin. This has shown integral membrane staining in all cells as a result of PS detectability. At low magnification, thymocytes undergoing apoptotic death are not randomly scattered but die in small groups. At higher magnification, specific membrane staining of the individual cells can clearly be visualized. Labelled Annexin V can be applied in flow cytometry, light microscopy and in both vital and fixed material by using appropriate protocols. Annexin V, an extension to the current available methods anticipates understanding of basic mechanisms that underlie apoptosis offering potential new targets that show an imbalance between cell proliferation and cell loss for therapeutic treatment of diseases. Annexin V labels necrotic cells membranes too. Thus, a control for membrane integrity of the PS-positive cells must be demonstrated (Heyder et al., 2003). Loss of membrane integrity is a pathogenomonic feature of necrosis, thus necrotic  C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy 

FLUORESCENT AND NONFLUORESCENT DNA STAINING TECHNIQUES

cells will stain with specific membrane-impermeant nucleic acid dyes such as PI, 7-aminoactinomycin D and trypan blue (Eijnde et al., 1997). In this way vital, apoptotic and dead cells can be discriminated on the basis of a double-labelling for Annexin V and PI, and analysed either by flow cytometry or fluorescence microscopy. The transfer of PS to the outside of the cell membrane will also permit the transport of certain dyes into the cell in a unidirectional manner. As the cell accumulates dye and shrinks in volume, the cell dye content becomes more concentrated and can be visualized with light microscopy. This dye-uptake bioassay works on cell cultures, not label necrotic cells and has a high level of sensitivity (can detect a single apoptotic cell; Cruchten, 2002). Apo2.7 antibody staining Apo2.7 is a protein confined to the mitochondrial membrane. It can be detected during early stages of apoptosis. It can be used to detect apoptosis via flow cytometry. Cells are first induced with apoptosis by giving specific treatment. Apoptotic cells are then incubated in 70% ethanol for 30 min. Nonspecific binding is blocked with Tris-NaCl-blocking reagent-buffer at room temperature for 45 min. Subsequently, cells are incubated with anti-Apo2.7 (2 mmol/L CaCl2 in TrisNaCl-blocking reagent) for 1 h, anti-mouse horse radish peroxidase for 1 h, tyramide signal amplification solution for 15 min, streptavidin FITC for 1 h and PI/RNase staining-buffer for 30 min. After each incubation step, cells are washed five times with Tris-NaCl-Tween-buffer. Positively stained cells obtained by the respective apoptosis assay are quantified and alterations of cell morphology are monitored by fluorescence microscopy. Fluorescence micrographs are taken using 450–490 nm excitations for fluorescence (Cevik & Dalkara, 2003). It is used to identify and distinguish apoptotic cells from that of the necrotic cells (Jinshun et al., 2003). As it is a mitochondrial membrane protein, it gets activated only at the time of apoptosis and binds specifically to the anti-Apo2.7 antibodies, thereby giving fluorescence at specific wavelength. Apo2.7 is conjugated with PECy5 and it gives florescence at 675 nm. Addition of propidium iodide to cells labelled with Apo2.7 is not recommended as signal overlapping will take place between PE and Apo2.7. The solution whenever is to be used should be warmed in a water bath to redissolve the precipitate formed.

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identification of cell differentiation and function. They bind noncovalently to carbohydrates and are readily purified from plant tissues (Leathem & Atkins, 1983). For detection of surface exposure of altered carbohydrates, cells are stained with the fluorescence-labelled lectins such as DSL-FITC, NPn-FITC and GSLII-FITC. Cells are incubated with 4–5 μg of labelled lectin for 30 min at 4°C. After adjustment to 500 μL PBS, fluorescence is measured by flow cytometry. Goat IgG antibody may be taken as a control (Zhang et al., 2002). Lectins are carbohydrate-binding proteins that possess various carbohydrate specificities (Leathem & Atkins, 2006). They are widely used in histology and cytology for many purposes such as the identification of carbohydrate moieties of membrane components (Lutsik et al., 1989), tumour cell destruction (Kawiak et al., 1988) and the induction of cellular growth and differentiation (Khopade et al., 1998). The abovementioned three lectins are not cytotoxic for many cell types and all of these detected more binding sites on late apoptotic cells when compared with early apoptotic and vital cells. A sensitive means of localizing and identifying glycoconjugates in tissue sections is to use the binding property of lectins conjugated to fluorescein or enzyme labels. NPn lectin staining specifically detects apoptotic events earlier than Annexin V stain. The best use of this dye is that it remains stable for at least 12 h facilitating everyday laboratory use. Quantitative analysis of lectin binding has to be performed before starting with the samples. The election of appropriate lectin specific to the sample should be done to avoid any unnecessary lectin interactions, which will provide nonspecific signals in the result (Lucas et al., 1999; Neu et al., 2001). Conclusion The fraction of apoptotic cells can be identified by morphological analysis due to various staining. The methodology seems valuable and significant for the detection of cellular anomalies. This review will help researchers to analyse the experimental set up of staining and can thus be recommended as standard methods especially for preevaluating studies. Conflict of interest Authors declare no conflict of interest. Acknowledgement

Lectin staining Lectins are potentially useful tools in histopathology for the identification of carbohydrates and distinguishing cells according to their type, differentiation or function. Lectins are simple to use on fresh tissue but fixation and processing sequesters glycoconjugates and dissolves out fat linked sugars (Freshney, 1987). Lectins are specific carbohydrate-binding proteins of nonimmume origin and of increasing value in  C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy 

We acknowledge DBT for their financial support.

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