overview of flow cytometric methods used in ...

OVERVIEW OF FLOW CYTOMETRIC METHODS USED IN POPULATION BIOLOGY

DNA Flow Cytometry with Plants The majority of cells in land plants and macroscopic algae are enclosed by rigid and impermeable cell walls and tightly embedded in complex three–dimensional tissues. These characteristics are not compatible with flow cytometric (FCM) analyses, which generally require a suspension of free particles. While free cells with walls have largely been disregarded due to methodological problems, FCM analysis of protoplasts (i.e. plant cells devoid of their walls) has been more successful (Doleˇzel et al. 2007). Protoplast–derived nuclei have also been assayed, but it was not until a breakthrough in nuclei extraction involving chopping fresh tissues (Galbraith et al. 1983) that FCM became a widely used technique in plants. The chopping method works well with the majority of plant species and tissue types (i.e. leaves, stems, sepal, petals, roots, and thalli). Seeds are usually as suitable as actively growing tissues, but histograms must be interpreted cautiously due to the presence of two nuclei types (embryo + endosperm) with different ploidy levels. FCM analysis of nuclear DNA content in pollen grains is more challenging and often requires substantial modification of standard protocols. Similarly, procedures have been optimized for handling and measurement of microscopic phytoplankton algae (see also “Flow Cytometry with Microorganisms”). Although use of fresh tissue clearly predominate in contemporary plant FCM, some analyses can also be performed on fixed specimens (e.g. Sgorbati et al. 1986, Jarret et al. 1995). In addition, recently there has been significant progress in the use of desiccated and/or frozen plant tissues (e.g. Nsabimana & Van Staden 2006, Suda & Tr´avn´ıˇcek 2006a,b). Recent articles dealing with DNA analysis of plant samples using FCM that are particularly worth reading are Doleˇzel & Bartoˇs (2005) and Greilhuber et al. (2007). Detailed step–by–step protocols are available in a comprehensive methodological compendium Current Protocols in Cytometry; Units 7.6 (analysis of fresh somatic plant tissues; Galbraith et al. 1997), 7.29 (analysis of dormant seeds; Sliwinska 2006), 7.30 (analysis of dehydrated plant tissues; Suda & Tr´avn´ıˇcek 2006b), and 11.12 (analysis of phytoplankton; Marie et al. 2000) deserve particular attention. Standard methods and relevant citations for selecting plant appropriate tissue, preserving samples, and isolating and staining nuclei are summarized below.

1) Selection of tissue Land plants a) Somatic tissue: • leaf laminas (mostly a default option) • other tissues with photosynthetic activity such as leaf stalks, young stems, sepals, cotyledons, etc. (Suda et al. 2005) 1

• fresh petals or tepals (Mishiba et al. 2000, De Schepper et al. 2001, Talent & Dickinson 2005); this tissue may represent an efficient substitute for conventional leaves in some recalcitrant species • young root tips (Obermayer et al. 2002) Tissues to be analyzed should be intact, young, devoid of parasites and disease symptoms; prominent vascular bundles in leaves should be removed before analysis. Etiolated parts (e.g. leaf bases of graminoids still hidden in sheaths) may in some cases yield superior results due to lower level of secondary metabolites. Plants grown in experimental conditions should be amply watered before tissue collection in order to achieve sufficient turgor, which fosters nuclei release. b) Seeds: • fully developed seeds collected at maturity (Matzk et al. 2000, Sliwinska et al. 2005) c) Pollen grains: • mature pollen collected from anthers just after anthesis (Van Tuyl et al. 1989, Bino et al. 1990, Pichot & El Maˆataoui 2000, Zhang et al. 1992, Mishiba et al. 2000, Stehlik et al. 2007). Aquatic algae • thalli of macroscopic algae (Ar Gall et al. 1996, Asensi et al. 2001) • whole bodies of microscopic algae (i.e. pico- and nanophytoplankton), either from cultures or natural conditions (LaJeunesse et al. 2005) • zoospores and other free cell types (Parrow & Burkholder 2002, Peters et al. 2004) • cysts (Kremp & Parrow 2006)

2) Sample preservation Methods for preserving plant samples have only recently received increased attention in part due to a burst of FCM applications in botanical research. The quality of FCM results obtained from preserved tissue may vary greatly among species. a) Short–term storage of somatic tissues: • bagging of leaf tissue with a moistened paper and storage at low temperatures (in refrigerator); allows sample storage from several days (in soft–leaf taxa) to a few weeks (in taxa with rigid leaves) b) Longer–term storage of somatic tissues/nuclei: • isolation of nuclei by chopping tissue and preservation in 30% glycerol at approx. -20 ◦ C (Chiatante et al. 1990, Hopping 1993). Stained nuclei have also been preserved this way. Fixed samples may be stored for up to 9 months.

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• rapid desiccation of plant material and storage at about -80 ◦ C (Suda & Tr´avn´ıˇcek 2006a,b). Both conventionally–prepared herbarium vouchers and silica–dry ˇ ˇ tissues may be successfully analyzed (Smarda et al. 2005, Smarda & Stanˇc´ık 2006, Sch¨onswetter et al. 2007). In mosses, dehydrated plant vouchers seem to be suitable even for genome size estimation in absolute terms (Voglmayr 2000). Successful FCM analysis has been reported in up to 12–year old specimens of vascular plants. • rapid freezing of tissue and storage at -70 ◦ C / -80 ◦ C (Nsabimana & Van Staden 2006; P. Kron & B.C. Husband, unpublished). At least 3–year storage is feasible. Long–term preservation of somatic tissue is often associated with a decrease in fluorescence intensity. In these cases, DNA content estimates should be restricted to relative, not absolute, measures. c) Seeds: • drying of mature seeds and storage in cold conditions (e.g. in refrigerator at 4 ◦ C). Maximum life–time with respect to FCM analyses should be experimentally determined, but it may likely reach several decades. Interestingly, FCM has been successful even with inviable seeds (Matzk 2007). d) Pollen: • drying of mature pollen grains and storage at room temperature (Bino et al. 1990) or in refrigerator (at 4 ◦ C) (Stehlik et al. 2007). Maximum storage period remains to be determined.

3) Nuclei isolation from different tissues No less than 27 different buffers for nuclei isolation (or simultaneous isolation and staining) have been developed. Greilhuber et al. (2007) summarized the chemical composition of ten of the most popular non–commercial buffers, which collectively account for about three quarters of published FCM papers. An exhaustive list of buffers, including original recipes, is available online at http://flower.web.ua.pt/ (as a part of the FLOWER database aimed at gathering methodological data relevant to plant DNA flow cytometry; see Loureiro et al. 2007). a) Somatic tissues of land plants and macroscopic thalli: • mechanical chopping or slicing fresh tissues using a sharp razor blade or a scalpel (the most common procedure introduced by Galbraith et al. 1983); works well also with bryophytes (Voglmayr 2000) and several macroscopic algae (Ar Gall et al. 1996, Asensi et al. 2001). A filtration column may enhance suspension quality in some recalcitrant species (Lee & Lin 2005). • chopping fixed plant tissues (Sgorbati et al. 1986) • grinding tissue with a glass rod while maintaining a continuous flow of extraction buffer (Chiatante et al. 1990)

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• homogenizing tissue with ceramic beads (P. Kron & B.C. Husband, unpublished) • enzymatic treatment of fixed cell suspension (Pfosser 1989) • shaking samples in microtubes, which contain free sharp metal strips (unpublished modification; allows synchronous preparation of many samples, as for example using a 96–well plate, and easy control of chopping intensity; T. Sharbel, pers. communication) Centrifuging nuclei may help to remove cellular debris and cytosolic compounds. b) Seeds: • chopping whole seeds using a sharp razor blade or scalpel (Matzk et al. 2000, Koperd´akov´a et al. 2004, Sliwinska 2006) • homogenizing seeds using a mortar or tissue homogenizer (discussed in Matzk 2007) • crushing seeds between two pieces of finely granulated sand paper (considered as optimal procedure by Matzk 2007) c) Pollen grains (isolation of nuclei or sperm cells): • chopping or mechanically crushing intact mature pollen (Van Tuyl et al. 1989, Bino et al. 1990, De Paepe et al. 1990, Mishiba et al. 2000, Pichot & El Maˆataoui 2000) • chopping germinating pollen (Pichot & El Maˆataoui 2000) • bursting hydrated pollen grains in unbuffered sucrose solution followed by centrifugation using discontinuous Percoll density gradients (Zhang et al. 1992) • ultrasonically disrupting pollen in suspension followed by nuclei purification using centrifugation (Pan et al. 2004) • freezing pollen overnight in a buffer and chopping the frozen suspension (Stehlik et al. 2007) d) Protoplasts: • chopping isolated protoplasts (Chen et al. 1994, Mishiba & Mii 2000) • lysing protoplasts in a hypotonic buffer plus detergent (H¨ ulgenhof et al. 1988, Ulrich & Ulrich 1991, Maciejewska et al. 1999) More complex protocols using protoplast–derived nuclei often yield histograms with less cellular debris than does the conventional chopping methodology (compare results in Ulrich & Ulrich 1991). e) Isolated algal cells: • lysing cells in a hypotonic buffer with detergent (Vaulot et al. 1994, Peters et al. 2004). Detailed methodology is provided by Marie et al. (2000).

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4) Cells and/or nuclei fixation Chemical fixation is usually avoided in FCM analyses of plants. When used, fixation increases permeability of cellular membranes for fluorochromes and, in case of alcohol– based fixatives, also removes chlorophyll and other autofluorescent pigments. The following procedures have been applied with some success. Fixation of: • isolated nuclei in 70% or 95% ethanol (Jarret et al. 1995) • isolated nuclei in NaCl:ethanol solution (H¨ ulgenhof et al. 1988) • cell suspension in ethanol:acetic acid (Pfosser 1989); a modified ethanol:acetic acid fixative has been used with plant protoplasts (Puite & Ten Broeke 1983) • leaf and root tissues in formaldehyde + Tris buffer solution (Sgorbati et al. 1986) • algal cells in methanol:acetic acid (LaJeunesse et al. 2005) • algal cells in paraformaldehyde (Marie et al. 2000)

5) Particle staining with DNA–selective fluorochromes Particles (mostly nuclei) can be stained with fluorochromes either simultaneously or after nuclei extraction. There are a variety of DNA–selective fluorochromes (Shapiro 2003), of which the most widely used in population studies are: • propidium iodide: intercalating fluorescent dye (absorption maximum 535 nm, fluorescence maximum 617 nm) used to estimate DNA content in absolute units (usually at final concentration 50-150 µg/ml). Binds also to double–stranded RNA; hence, samples must be treated with RNase (final concentration 50 µg/ml). • DAPI: AT–selective fluorescent dye (absorption maximum 357 nm, fluorescence maximum 451 nm), which provides estimates of relative DNA content with high level of resolution. Usually used at final concentration 4 µg/ml. Recommended concentrations and optical properties of other fluorescent dyes applied in aquatic FCM are given by Marie et al. (2000).

6) Other methodological considerations a) Instrumentation: Standard biomedical flow cytometers are routinely used in plant biology, although instruments adapted for plant samples may yield superior results. In particular, special instrument design is required for phytoplankton (Dubelaar et al. 2007) and occasionally also for protoplast (Galbraith 2007) analyses. Details on flow cytometer operation and set-up can be found in Galbraith et al. (1997). • propidium iodide–stained samples are optimally analysed on instruments equipped with a green solid–state laser (532 nm); excitation with conventional argon– ion turquoise laser (488 nm) usually provides less satisfactory results. Optionally, an arc lamp may be used together with a high–aperture objective (Temsch et al. 2001). 5

• DAPI–stained samples are mostly excited by UV bands emitted from mercury arc lamp; UV lasers are only rarely available in bench–top instruments. b) General methodological guidelines: • standardization and standard selection Accurate interpretation of FCM results requires the use of a reference standard with known characteristics (e.g. ploidy level, genome size). This issue is detailed by Doleˇzel & Bartoˇs (2005) and Greilhuber et al. (2007). DNA ploidy results should ideally be calibrated using chromosome counts (Suda et al. 2007). • quality requirements and trouble–shooting Quality standards for estimating genome size and ploidy, together with methodological guidelines are summarized by Greilhuber et al. (2007) and Suda et al. (2007), respectively. The former paper discusses the potential negative effects of secondary metabolites on DNA staining. Suda (2004) provides some tips for analysis of recalcitrant vascular plant species. • resolution threshold The resolution of FCM estimates of single optical parameters (e.g. DNA content) largely depends on the coefficients of variation (CV) of the histogram peaks. As a rule of thumb, two peaks only be distinguished if their mean difference in simultaneous FCM runs are larger than 2 x CV of each peak. In particular, histograms with small variances are required to distinguish euploids from near–euploid aneuploids (Benson & Braylan 1994, Roux et al. 2003). Aneuploids are detected by using an euploid as an internal standard, and identifying aneuploids by the presence of two distinct G0/G1 peaks (Shankey et al. 1993 (clinical practice), Pfosser et al. 1995, Schween et al. 2005); or by a measured deviation in absolute DNA content from known euploids (e.g. Bashir et al. 1993, Baebler et al. 2005).

DNA Flow Cytometry with Animals In contrast to plants, intact animal bodies contain free cells, such as blood / hemolymph cells and, in the case of males, sperm cells, which require little preparation for FCM analyses. The absence of a rigid cell wall is another distinct feature with significant methodological implications (e.g. easier cell lysis). In addition, while the majority of plant protocols involve fresh tissues, animal samples are routinely fixed and/or frozen before FCM acquisitions.

1) Selection of tissue When nucleated, blood erythrocytes are the most common cell type used for DNA content estimates in animals (vertebrates). Along with their ease of collection, other advantages include low RNA content and the lack of secondary metabolites, which may interfere with DNA staining. In addition, sampling is non–lethal, at least in vertebrates with a reasonable body size.

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Invertebrates Whole bodies or body parts (e.g. heads) are most often used although there is the potential for staining inhibitors to occur. • liver cells (e.g. in mollusc; Vinogradov 1998a) • hemocytes (e.g. in mollusc; Gagnaire et al. 2006); may also be collected non–lethally as for example after a limb abscission in spiders (J. Suda et al., unpublished) • detached heads (e.g. in insects; Nardon et al. 2003) • whole bodies (e.g. in small crustaceans and worms; Korpelainen et al. 1997, Bennett et al. 2003) Vertebrates (except mammals) • blood cells (erythrocytes) collected either from peripheral blood (Birstein et al. 1993, De Vita et al. 1994, Cavallo et al. 1997, Lizana et al. 2000, Borkin et al. 2001, Krishan et al. 2005) or from the heart after animal anesthetization or sacrifice (Borkin et al. 2001, Hickey & Clements 2005, Ramsden et al. 2006). The most common protocol with virtually universal application (fishes, amphibians, reptiles, and birds). • whole fin tissues and/or fin epithelium cells in fishes (Lamatsch et al. 2000, Morishima et al. 2002) • pulp feather cells in birds (Tiersch & Mumme 1992) • hepatocytes (Vinogradov & Borkin 1993) • bone marrow cells (Vinogradov & Borkin 1993) • kidney cells (Lockwood & Bickham 1991) • testicular and/or epididymal tissue (Bickham et al. 1993) • retinal cells (Ciudad et al. 2002) • sperm cells (Alves et al. 1999, Linhart et al. 2006) • whole body (e.g. in frog tadpoles; Freeman & Rayburn 2004) Mammals Because mammalian red blood cells are enucleate, other cell types have to be collected for DNA flow cytometric analyses, including: • spleenocytes (Burton et al. 1989, Vinogradov 1998b, Borkin et al. 2001) • hepatocytes (Vindeløv et al. 1983a) • thymocytes (Vinogradov & Borkin 1993) • bone marrow (Otto et al. 1981) • leukocytes (Vinogradov 1994) • sperm cells (Pinkel et al. 1982) 7

2) Collection of tissue samples Blood samples are usually directly mixed with a buffer or free–cells are obtained after homogenization of solid tissue in the buffer. Many buffers conventionally used with plant cells yield good results with animal samples and vice versa (see for example Bennett et al. 2003). The most widely used buffers in animal flow cytometry are: • phosphate buffer saline + EDTA (Tiersch et al. 1989, De Vita et al. 1994, Cavallo et al. 1997, Vinogradov 1998a,b, Borkin et al. 2001, Freeman & Rayburn 2004) • citrate buffer (Otto 1992, Lamatsch et al. 2000) • sodium citrate buffer + trisodium citrate + dimethylsulfoxide (Vindeløv et al. 1983a, Lizana et al. 2000, Ciudad et al. 2002) • Hanks balanced salt solution (Burton et al. 1989, Vinogradov & Borkin 1993) • ACD (acid–citrate–dextrose) anticoagulant solution (Tiersch et al. 1989, Tiersch & Mumme 1992) In most cases, cells suspended in the buffer are refrigerated immediately after collection. Occasionally, intact tissue may be frozen before homogenization (e.g. Burton et al. 1989). Temperatures for sample storage vary from 4 ◦ C (De Vita et al. 1994; up to 4–week storage was possible) to -20 ◦ C (Lizana et al. 2000, Ciudad et al. 2002, Krishan et al. 2005) and -80 ◦ C (Vindeløv et al. 1983a, Tiersch et al. 1989, Alves et al. 1999).

3) Cell fixation Animal cells are often fixed to achieve higher stability and improve permeability of the plasma membrane to fluorochromes (DNA dyes do not readily cross intact membranes). Alcohol–based fixatives are most readily used, including: • 45-50% ethanol at 4 ◦ C or -20 ◦ C (Birstein et al. 1993, Freeman & Rayburn 2004) • 70% ethanol at 4 ◦ C (Otto et al. 1981, Lamatsch et al. 2000); storage for up to several months has been reported (Otto 1992) • 100% methanol at -20 ◦ C (Hickey & Clements 2005) • 10% phosphate buffered formalin at 4 ◦ C (Pinkel et al. 1982); NB: formalin fixation was not recommended by other researchers (e.g. Otto 1992).

4) Nuclei isolation a) use of non–ionic detergents: The most straightforward procedure for extracting nuclei involves addition of a non– ionic detergent (Triton X–100, Nonidet P–40, Tween 20) to the cell suspension. These agents may be used alone (Vinogradov & Borkin 1993, Vinogradov 1998a, Borkin et al. 2001) or, more commonly, as a component of nuclear isolation buffers, such as: • sodium citrate buffers (Tiersch et al. 1989, Tiersch & Mumme 1992, De Vita et al. 1994, Cavallo et al. 1997, Lamatsch et al. 2000, Krishan et al. 2005) 8

• Tris–based buffers (Birstein et al. 1993, Korpelainen et al. 1997) b) detergent–trypsin method: • extracting nuclei from unfixed cells using a non–ionic detergent and digestion of cytoplasm by trypsination (Vindeløv et al. 1983a, Lizana et al. 2000, Ramirez et al. 2001, Ciudad et al. 2002); more sophisticated procedure, which often improves resolution of FCM analyses.

5) Particle staining with DNA–selective fluorochromes Isolated nuclei are usually stained at the same time as they are extracted (i.e. by adding of DNA–selective dyes into the lysis buffers). In some cases, separate staining buffers have been prepared, including: • trisodium citrate–based buffer (Vindeløv et al. 1983a, Lamatsch et al. 2000, Lizana et al. 2000, Ramirez et al. 2001, Ciudad et al. 2002) • sodium hydrogen phosphate solution (Otto et al. 1981, Otto 1992) • Tris–HCl buffer (Burton et al. 1989) As with plant samples, propidium iodide is the most frequent fluorescent dye used in animal flow cytometry. Other popular fluorochromes include: • DAPI (Lamatsch et al. 2000, Morishima et al. 2002, Krishan et al. 2005) • ethidium bromide (Korpelainen et al. 1997) • Hoechst 33258 (Vinogradov & Borkin 1993, Vinogradov 1994) • olivomycin (Vinogradov 1998a,b, Borkin et al. 2001)

6) Other methodological considerations A number of organisms have been proposed as internal reference standards for FCM analyses (Tiersch et al. 1989), the most commonly used are: • trout red blood cells (Jakobsen 1983, Vindeløv et al. 1983b) • human lymphocytes (Jakobsen 1983) • chicken red blood cells (Vindeløv et al. 1983b); NB: selection of chicken cells as reference standard has recently been seriously discouraged due to their genome size instability. One–parameter (i.e. fluorescence values) is generally recorded and the results are expressed as one–dimensional histograms.

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Flow Cytometry with Microorganisms FCM protocols for microoorganisms have been well reviewed for different taxa: planktonic bacteria (Gasol & Del Giorgio 2000), phytoplankton (Marie et al. 2000, Dubelaar et al. 2007), aquatic communities in general (special issues of Cytometry in 1989 and 2001, and Scientia Marina in 2000; Vives-Rego et al. 2000), viruses (Brussaard et al. 2000), and microorganisms in general (Porter et al. 1997, Gruden et al. 2004). Here we briefly outline some of the recurrent themes in microorganism FCM methodology. In many cases, whole intact cells and/or cell colonies are analysed, so sample pre– treatment beyond filtering, such as isolation of nuclei from solid tissues or protoplast preparation, is not necessary. In some cases (e.g. phytoplankton), classification can proceed on the basis of autofluorescence and scatter properties (i.e. without staining), but the general trend is towards increasingly complex staining and labeling procedures (e.g. FISH, antigenic, and species–specific nucleic acid labeling) designed for the simultaneous study of multiple taxa in mixed samples (Porter et al. 1997, Vives-Rego et al. 2000, Gruden et al. 2004, Dubelaar et al. 2007). This coincides with a general trend to couple FCM with other molecular techniques, either to expand the discriminatory power of FCM as mentioned, or to complement FCM with related data on other community members or functions (e.g. Larsen et al. 2004, Countway & Caron 2006). In a similar way, sorting by FCM may be used to isolate organisms that can then be better studied by other methods (Vives-Rego et al. 2000). Application of FCM to microoorganisms has involved equipment modifications, primarily to deal with organism size, low fluorescence, volume control, and portability (Gasol & Del Giorgio 2000, Dubelaar et al. 2007). New staining protocols have been developed to deal with unique aspects of microorganisms, such as the impermeable walls of spores and cysts (e.g. Kremp & Parrow 2006). The need to discriminate among numerous components of complex mixed samples, which has no parallel in the study of other organisms, has driven new analytical approaches, including multivariate statistical analyses, the use of particle shape measures, and the potential use of “neural networks” (Porter et al. 1997, Vives-Rego et al. 2000, Gruden et al. 2004).

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