Because of their extensive availability and the wade spectrum of carbohy drates that may be specifically bound, lectms have become essenttal reagents for detection and quantttatton of glycoconlugates in solutton and m cell sur faces, identification and separation of cells, and functional studies based on membrane properties (Z,2) Flow cytometry is an analyttcal process m which several physical and/or chemical properties of mdivtdual cells or particles may be measured at a high velocity. Because of these features, flow cytometry 1s used extensively m basic and climcal research (3). In the context of glycoconjugate analysis, the apph cation of flow cytometry and fluorescent lectms started m the early 1970s (4). Although the advent of MAb decreased the use of lectms as tools for cell tden trfication, there are still a number of interesting flow cytometric apphcatlons which involve fluorescent lectm bmdmg, especially in multiparametric combt nation with other structural and functional cellular features (.5,6)
Home Lectin Methods and Protocols Protocol Analysis of Subcellular Components by Fluorescent-Lectin Binding and Flow Cytometry Download book PDF Download book EPUB Analysis of Subcellular Components by Fluorescent-Lectin Binding and Flow Cytometry Rosa M. Guasch & José-Enrique O’Connor Protocol 1807 Accesses Part of the Methods in Molecular Medicine™ book series (MIMM,volume 9) Abstract Because of their extensive availability and the wide spectrum of carbohydrates that may be specifically bound, lectins have become essential reagents for detection and quantitation of glycoconjugates in solution and in cell surfaces, identification and separation of cells, and functional studies based on membrane properties (1,2) Keywords Flow Cytometric Analysis Lectin Binding Isolation Buffer List Mode Data Specific Lectin These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves. Access provided by Universitat de València Download protocol PDF Springer Nature is developing a new tool to find and evaluate Protocols. Learn more 1 Introduction Because of their extensive availability and the wide spectrum of carbohydrates that may be specifically bound, lectins have become essential reagents for detection and quantitation of glycoconjugates in solution and in cell surfaces, identification and separation of cells, and functional studies based on membrane properties (1,2) Flow cytometry is an analytical process in which several physical and/or chemical properties of individual cells or particles may be measured at a high velocity. Because of these features, flow cytometry is used extensively in basic and clinical research (3). In the context of glycoconjugate analysis, the application of flow cytometry and fluorescent lectins started in the early 1970s (4). Although the advent of MAb decreased the use of lectins as tools for cell identification, there are still a number of interesting flow cytometric applications which involve fluorescent lectin binding, especially in multiparametric combination with other structural and functional cellular features (5,6). 1.1 Application of Fluorescent Lectins to Subcellular Analysis by Flow Cytometry Intracellular membrane systems, such as endoplasmic reticulum, Golgi complex, mitochondria, glyoxysomes and chloroplasts contain accessible carbohydrates bound to lipids or proteins (7,8). Such glycoconjugates are thought to play important roles in maintaining the structure and function of organelle membranes. Thus, glycosylated proteins may provide conformational stability, surface charge, water-binding capacity, and resistance to proteases (9), whereas glycolipids are involved in the regulation of membrane fluidity and shape, ion binding, and membrane interactions (10). In spite of such interesting properties, there have been only limited attempts to apply fluorescent lectins and flow cytometry to the analysis of intracellular glycoconjugates. This lack of information reflects in part the methodological diffculties in the flow cytometric analysis of some intracellular structures. Thus, subcellular analysis in whole cells may require careful fixation and permeabilization procedures when relatively large (e.g., conjugated lectins or antibodies) or nonpermeable fluorescent molecules are used as reagents (11). On the other hand, the use of isolated subcellular particles demands proper instrumental sensitivity and resolution to establish functional or structural criteria to distinguish single subcellular elements from background noise, as will be pointed out later in this chapter. Nevertheless, flow cytometric analysis of subcellular compartments may be performed satisfactorily with most of the currently available instruments (7,12). Thus, Petit and coworkers (7,13) have been pioneers in the application of fluorescent lectins to the flow cytometric analysis of isolated subcellular elements, namely mitochondria, chloroplasts, and thylakoids from different plant tissues. More recently, we have applied flow cytometry to the detection and quantitation of the binding of several glycoconjugate specific lectins to isolated cis and trans Golgi fractions from rat liver (14,15). 1.2 Application of Fluorescent Lectins to the Analysis of Isolated Golgi Elements The maturation and distribution of membrane and secretory glycoproteins involve the transit of newly synthesized proteins through the Golgi complex (16), which is organized into three functionally distinct regions, the cis Golgi network, the medial Golgi, and the trans Golgi network (17). The cis and trans Golgi networks constitute the entry and exit faces of the stack, respectively, and have morphological and functional differences because each one includes distinct processing enzymes (17). A substantial part of the current knowledge of the functions of the Golgi complex and the heterogeneity of glycosylation events within its subcompartments has arisen from cytochemical and biochemical analysis of lectin binding to specific carbohydrates (18,19). In view of the capabilities of flow cytometry, we have extended these studies to the detection and quantitation of the binding of several fluorescein conjugated lectins, namely concanavalin A (Con A), wheatgerm agglutinin (WGA), Ulex europaeus agglutinin (UEA) and Arachis hypogaea agglutinin (PNA) to isolated cis and trans Golgi fractions from the liver of normal and ethanol treated rats (14,15). Our results showed that flow cytometric analysis, using low-power, air cooled laser, is adequate to study carbohydrates present in isolated Golgi membranes. Thus, the use of specific lectins allows the determination of. the intensity of specific binding of different lectins to each Golgi subcompartment, the percentage of elements that bind different lectins specifically within the same subcompartment; and the intensity and percentage of lectin binding when comparing cis and trans fractions. All these parameters were affected to different extents by chronic exposure to ethanol (14,15), thus indicating the feasibility of applying the technique described here to detect alterations in the pathways of glycoconjugate metabolism. 2 Materials 2.1 Purification and Isolation of Golgi Subfractions 1. Isolation buffer 1 0.25M sucrose, 10 mM Tris-HCl, pH 2. Isolation buffer 2 1 35M sucrose 3. Discontinuous sucrose density gradient: 2.00, 1 35, 1 15, 0 86, and 0 25M sucrose. 7 5 mL of each concentration are used for each single tube in step 7 of Section 3 1 4. Potter Elvehjem homogenizer with Teflon® pestle 5. Beckman L5-65 ultracentrifuge with Beckman SW 27 rotor (Fullerton, CA) 2.2 Lectin Preparation 1 Fluorescent lectins, conjugated to fluorescein isothiocyanate (FITC, green fluorescing) or to tetramethylrhodamine isothiocyanate (TRITC, red fluorescing) (Sigma, St. Louis, MO) 2 lectin binding inhibitory sugars: specific to the lectins selected for the study (Sigma) (see Note 1 and 2) 2.3 Flow Cytometry 1 Dulbecco phosphate-buffered saline (DPBS) with calcium and magnesium. 2 Any laser or mercury lamp based flow cytometer, able to provide 488 nm excitation light (blue) and to detect forward angle light scatter, right angle light scatter, and green or red fluorescence 3 Methods 3.1 Isolation of Golgi Cis and Trans Fractions 1. Golgi cis and trans subcompartments may be isolated from the liver of rats (150–200 g) following a modification (20) of the procedure of Bretz et al. (21), as follows. 2. Sacrifice the animals by cervical dislocation, remove their livers, wash them briefly in ice-cold isolation buffer 1, and weigh them. 3. Homogenize the livers in ice-cold isolation buffer 4. Dilute the homogenate to 20% (w/v) concentration with isolation buffer 1 and centrifuge for 10 min at 10,000g at 4°C. 5. Separate the resulting supernatant and centrifuge for 90 min at 105,000g at 4°C to obtain a microsomal pellet. 6. Resuspend the pellet in 7 5 mL of isolation buffer 7. Layer the sample under a discontinuous sucrose density gradient (7.5 mL each of 2 00, 1 35, 1 15, 0 86. and 0 25M sucrose) prepared in polyallomer tubes and centrifuge it at 82,000g for 3 h at 4°C with a Beckman SW 27 rotor. 8. Collect separately the material floating to the 0 25/0 86M interface (trans Golgi fraction) and to the 0 86/1 5M interface (cis Golgi fraction). Keep stored at 4°C and analyze before 24 h. 9. Dilute Golgi fractions in DPBS to an equivalent to 100µg protein/ml for flow cytometric analysis (see Note 3–6) 3.2 Flow Cytometry 3.2.1 Instrumental Settings and Parameter Selection 1 Excite the FITC- or TRITC-conjugated lectins with argon-ion laser or mercury lamp, both tuned at 488 nm. 2 Use a filter setting homologous to the one suggested here for the photomultiplier (PMT) arrangement of the Coulter EPICS Elite Cell Sorter (it may vary depending on the instrument) 488 nm dichroic +488 band pass (in PMT1, right angle light scatter) +488 nm blocking +550 nm dichroic +525 nm band pass (in PMT2, green fluorescence) +575 nm dichroic +610 long pass (in PMT3, red fluorescence) (see Note 7). 3 Estimate particle morphology by logarithmic amplification of forward angle light scatter (proportional to particle size) and right angle light scatter (dependent on particle texture). Quantitate lectin binding by logarithmic amplification of green (FITC) or red (TRITC) fluorescence. 3.2.2 Discrimination of Golgi Elements from Background Noise 1 Specific selection of Golgi particles based on light scatter is necessary, prior to the analysis of fluorescence. Because of their small size, these particles are not easily discriminated from background noise. 2 Incubate with a fluorescent lectin (e g, FITC-Con A, 200µg/mL, for 1 h at 37°C in DPBS). 3 Perform the backgating procedure as shown in Fig. 1. Fig. 1. figure 1 Flow cytometric detection of isolated Golgi elements. (A) Biparametric histogram of forward angle light scatter (LFALS) vs right angle light scatter (L9OLS) showing the population of Golgi elements and background noise; (B) superimposition of histograms of green (LIGFL) autofluorescence (AFL) and FITC Con A fluorescence of Golgi fractions, showing the lectin binding population considered for backgating, included within a linear cursor; (C) biparametric histogram of LFALS vs L9OLS gated on LIGFL showing the cytometric morphological properties only of the elements included within the cursor in (B), which are able to bind FITC Con A and, thus, selected for fluorescence analysis; and (D) biparametric histogram of LFALS vs LIGFL showing that the morphological region selected indudes positive and negative Con A binding fractions in the Golgi population separated from noise. Thus, the morphological region shown in (C) is selected within an electronic bitmap and all the fluorescence measurements restricted to these elements. The described procedure should be performed for each individual preparation. Full size image 3.2.3 Titration of Lectin-Specific Binding 1 To establish the optimal conditions for lectin binding, incubate Golgi fractions (100µg protein/ml) for 1 h at room temperature with increasing concentrations of each conjugated lectin. Following incubation with the lectin, centrifuge each sample for 10 min at 15,000g and resuspend in 1 mL of DPBS, before flow cytometric analysis. 2 Nonspecific binding is estimated in aliquots preincubated for 1 h with excess of adequate inhibitory sugars. 3 Specific lectin binding is calculated as the difference between the mean fluorescence intensities of total (samples incubated with fluorescent lectin in the absence of inhibitor) and nonspecific binding (samples incubated with fluorescent lectin in the presence of inhibitor) in monoparametric histograms as shown in Fig 2. When no difference in the mean fluorescence of total and nonspecific binding is observed, Overton substraction of histograms must be applied (22). 4 For performing flow cytometric analysis with lectins, select the concentration giving the maximal degree of specific binding in the titration curve, as exemplified in Fig 3. As a guide, Table 1 shows the concentrations of lectins and competitive sugars that we found optimal in our experiments with isolated Golgi fractions. Fig. 2. figure 2 Representative histograms of the binding of Con A to isolated cis (A) and trans (B) Golgi fractions. Each panel is a superimposition of three histograms autofluorescence (AFL) of unlabeled fractions, nonspecific binding (Con A+I) of the lectin in samples preincubated with the presence of 0 01M α-D-methylmannose, and total binding (Con A) of the lectin without preincubation with the inhibitor Con A binding shows an homogeneous population that shifts in the presence of the inhibitor. Full size image Fig. 3. figure 3 Characterization of the binding of FITC WGA to isolated Golgi elements. Total and nonspecific binding were determined in the absence and presence of the sugar inhibitor, respectively, as shown in Fig 2. Specific binding was calculated by substracting the mean value of nonspecific binding from that of total binding. Each point represents the mean of four experiments. In this example, the optimal lectin concentration was shown to be 50µg/mL ▪, total binding, ▴, specific binding, ⊗, nonspecific binding. Full size image Table 1 Optimal Concentrations of Lectins and Inhibitors Full size table 4 Notes 1. Handle lectins with care, since some of them may be mitogenic or toxic. 2. Perform all the steps involving fluorescent lectins in the dark or under subdued light. Keep the stock solutions protected with aluminium foil. 3. The purity of the Golgi preparations may be assessed by biochemical analysis in the homogenate and in the fractions of the following enzyme markers 5′-nucleotidase (plasma membrane), glucose 6 phosphatase (endoplasmic reticulum), acid phosphatase (lysosomes), alcohol dehydrogenase (cytosol), glutamate dehydrogenase (mitochondria) (20). 4. The relative enrichment in cis and trans Golgi in the fractions may be quantified by assaying galactosyl transferase, a specific marker of trans Golgi (20). 5 The cytochemical detection of thiamine pyrophosphatase, a trans Golgi marker, may be used to assess the purity of fractions by electron microscopy (20). 6 Avoid freezing and thawing the preparations of Golgi subfractions, since this may disrupt the isolated vesicles and increase very much the background noise. 7. The flow cytometer settings recommended here have been tested and shown adequate for the range of Coulter EPICS instruments using low power (15 mW), air cooled argon laser and quartz flow chambers (EPICS Elite, Profile II and XL). Petit et al (7,13) have performed then flow cytometric analysis of lectin binding to isolated plant mitochondria, chloroplasts, and thylakoids using an EPICS V with open flow chamber, which demands a higher laser power (400 mW, ref 13). 8 To provide an adequate data rate (400–500 events/s), while avoiding particle coincidence during the flow cytometric analysis, a dilution of subcellular fractions equivalent to about 100µg protein/ml is recommended. 9 When possible, store flow cytometric data in the form of list mode files, since this allows one to reanalyze data off line as desired, thus permitting gate relocation, different parameter combination, and so on. 10 All current flow cytometers have more than adequate software for performing on-line and off-line data analysis and there are several commercial sources for off-line analysis on MS-DOS, Windows, and Macintosh systems. 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