And Paraffin Embedded TissueProtocol

In formalin-fixed and paraffin-embedded tissue material, the DNA is trapped in a relatively strong matrix of cross-linked protein. If the tissue material is fixed at neutral pH for not longer than 24 hr, the DNA and proteins are internally cross-linked to a limited extent. This protocol describes such a cross-linking procedure. Incubation in formic acid H2O2 removes protein prior to permeabilization with pepsin, improving nuclear isolation and permeabilization. Following permeabilization,...

Preparation Of Dna Fibers From Fibroblasts By The Halo Technique

One of the oldest techniques used for DNA fiber-FISH mapping is called the halo preparation technique (Wiegant et al., 1992), a modified version of the technique described by Vogelstein et al. (1980). Cells growing on a glass slide are lysed in a buffer containing a detergent. This is then followed by high-salt washes to remove histones from the chromatin. The DNA in such nuclei consists of loops anchored to the nuclear matrix in a form having a high degree of negative supercoiling. Next, the...

TrisEDTA buffer pH

Dissolve 3.7 g Tris base and 0.121 g disodium EDTA in < 1 liter distilled water. Adjust pH to 8.0 at 4 C, using 1 N HCl, bring up to 1 liter with water. Filter solution using a 0.22- m filter and store up to 1 month at 4 C. Figure 11.13.3 (A) DNA two-parameter histogram (green fluorescence signal area versus green fluorescence signal height) for Zygosaccharomyces bailii (ISA 1307) (n 30,000) after SYBR Green I staining. This distribution shows an apparent high synchrony of cells in G2 M...

Info

Typically required for PNA RNA hybridization in comparison to DNA RNA hybridization. FISH assays can be made more specific by adjusting the wash step conditions. After hybridization, it is important to remove all the PNA that may be partially hybridized to nontarget sequences. This can be achieved in the wash step by increasing the temperature, pH, and or denaturant concentration to increase the binding stringency. Under these conditions, the tolerance for mismatches will be low, and the...

Support Protocol 1

The primary monoclonal antibody (MAb) and the detection reagents must be titrated to determine optimal concentrations. This is done by performing staining and analysis with varied concentrations of MAb and detection reagents. Thus, reagents and specific methods are as described (see Basic Protocol). Modifications for titration are described below. Note that the use of an isotype-matched negative control is an essential but not necessarily adequate control, because one antibody can give higher...

Commentary Background Information

In recent years green fluorescent protein (GFP), cloned from the bioluminescent jellyfish Aequorea victoria, has become a widely applied reporter of gene expression and protein localization. The existence of green fluorescent proteins had been suggested around 1970 when discrepancies between the in vivo and in vitro bioluminescent spectra of coelenterates implied an energy-transfer phenomenon from the initial calcium-activated phosphoprotein (Morin and Hastings, 1971a,b Wampler et al., 1971)....

Microscope Hardware

Because digital fluorescence microscopy (DFM) often aims at quantitative analysis of fluorescence images, requirements with respect to quality of the microscope hardware are high. Quantitative analysis will reveal errors such as chromatic or spheric aberration of lenses, uneven illumination of the object, unwanted excitation light (stray light), and autofluorescence of optics and filters in an often frustrating way. Because the light-collection efficiency of microscopes has increased...

Critical Parameters

The assay described was originally designed to be performed in a clinical laboratory within 6 hr. In this regard, it is important to note that although the percentage of cells that will up-regulate their surface expression of CD40L (following in vitro activation with PMA and ionomycin, as described herein) stabilizes after 3 hr, the level of expression of CD40Lper cell (measured as the mean or median fluorescent channel) continues to rise after a 5-hr incubation (Fig. 6.7.3). Therefore, if...

Background Information

A majority of agriculturally important plant species have a complex nuclear genome that makes gene mapping and isolation very difficult. This daunting task may be simplified by dividing the genome into well-defined parts, e.g., chromosomes. Generally, individual chromosomes can be isolated either by chromosome microdissection or by flow sorting. The advantage of flow cytometry is that chromosomes can be sorted in large numbers. Flow analysis and sorting of mitotic chromosomes (flow...

Dna With Acridine Orangeprotocol

In this protocol cells are fixed in 70 ethanol (apoptotic cells are briefly prefixed in 1 formaldehyde). In addition to fixation, ethanol permeabilizes cells, making DNA accessible to the dye. The cells are then centrifuged to remove the fixative, treated with RNase, and then with 0.1 M HCl to denature DNA, and stained with acridine orange (AO) at pH 2.6. The low pH of the staining solution precludes DNA renaturation, which would otherwise occur. AO differentially stains the nondenatured...

Cleaning

While a detailed treatment of cleaning supplies and methods is beyond the scope of this section, some discussion is warranted. Consult your microscope's manufacturer or local dealer for any booklets or written materials that might be available. Many service dealers can provide expert advice and tips, as well as proper cleaning supplies, so don't hesitate to consult them. Selection of the proper cleaning fluid is important, and there are many opinions about which cleaners and solvents are safe...

Multiplicity

The multiplicity of a FISH procedure is defined as the number of DNA targets that can be distinguished on the basis of optical properties, usually fluorescence color. In the simplest application of multiplicity, different fluoro-chromes that are spectrally well separated are attached to separate probes either directly or indirectly. For the visible part of the electromagnetic spectrum, blue, green, and red fluorescent dyes are available, permitting a multiplicity of 3 for visual observation of...

Immunophenotyping Of Peripheral Blood Leukocytes

Whole blood is collected using a tube containing EDTA as the anticoagulant. Whereas a conventional blood draw is 7 to 10 ml or more, these protocols require only 500 l or less. The blood cells in suspension are stained with three monoclonal antibodies that are directly conjugated to different fluorochromes. The erythrocytes are lysed, and the sample is centrifuged and resuspended in PBS. The stained leukocytes are then mounted on conventional uncoated glass microscope slides and air dried....

Flfcm Instrumentation And Signal Processing

Ramsey and Hieftje (1983) have made a detailed study of the relative merits of various modulation and signal-processing schemes based on signal-to-noise ratio in shot noise limited luminescence measurement systems. Their conclusions were that high signal-to-noise ratios are achieved by (1) maximizing the measured radiant photon flux and observation time, (2) maximizing the ratio of the experimental bandwidth to the signal detector bandwidth, and (3) minimizing the number of frequency components...

Nfat

Although it is known that FcyRIII cross-linking activates many molecules and signaling pathways, less has been described about the mechanisms by which these pathways can result in transcriptional control of a variety of genes. One transcription factor, which was shown to be activated in NK cells after FcyRIII cross-linking, is nuclear factor of activated T cells (NFAT Aramburu et al., 1995). Cytoplasmic NFAT undergoes dephosphorylation by the phosphatase calcineurin. This dephosphoryla-tion...

Surface Antigens

The primary mouse monoclonal antibody of the IgG1 or IgG2a isotype must be used at saturating concentration. Ideally, this concentration will be below 5 g ml (final). Additional Materials (also see Alternate Protocol 3) Unconjugated primary antibody Cellular sample containing both negative and positive cells for the antigen detected by the unconjugated primary antibody FITC-conjugated GAM antibody 1. Prepare a series of 1 1 serial dilutions (i.e., 1 1, 1 2, 1 4, 1 8, etc.) of each unconju-gated...

Dna Fragmentation Detection Of Cells With Fractional Subg1 Dna Content Using Dapi

Cellular DNA may be stained with other fluorochromes instead of PI, and other cell constituents may be counterstained in addition to DNA. The following is the procedure used to stain DNA with DAPI. This protocol requires a UV laser. Additional Materials (also see Basic Protocol 6) DNA staining solution with DAPI (see recipe) Flow cytometer equipped with UV excitation and filter for collection of blue fluorescence 1. Follow Basic Protocol 6, steps 1 to 4. Then, suspend the cell pellet in 1 ml...

Literature Cited

Digital Image Processing Principles and Applications. John Wiley & Sons, New York. Castleman, K. R. 1996. Digital Image Processing. Prentice-Hall, Englewood Cliffs, N. J. Gonzalez, R. C. and Woods, R. E. 1992. Digital Image Processing. Addison-Wesley, Reading, Mass. Jain, A. K. 1989. Fundamentals of Digital Image Processing. Prentice-Hall, Englewood Cliffs, N. J. Pratt, W. K. 1991. Digital Image Processing, 2nd ed. John Wiley & Sons, New York. Russ, J. C. 1995. The...

Postprocessing Gates Digital Gating

Digital gating occurs after the analog stretch pulse has been converted to a digital value. The digital value can then be used by itself or combined with the digital values of other parameters and applied to a digital gating circuit. The simplest form of a digital gating circuit is a region. For a given parameter, two comparisons are made by digital comparators to determine whether the digital value is within the set limits. If two parameters are involved, then a bitmap is used. This is a...

Epitope Titering

The previous procedures (see Basic Protocols 1 and 2 and Alternate Protocols 1 and 2) determine the antibody titer at a constant epitope concentration. The working titer of any antibody is also dependent upon the concentration of epitopes. The optimal antibody concentration required to saturate cells having 105 epitopes is different from that required to saturate cells having 104 epitopes, and both cell types can be present in the same suspension. To determine this titer, the antibody...

Detection Of Tissue Transglutaminase Activation By Cell Resistance To Detergents

Extensive protein crosslinking takes place during apoptosis. The ubiquitous transglutami-nase TGase 2 (also called tissue transglutaminase tTGase) was identified as the enzyme responsible for this reaction (Fesus et al., 1987 Melino and Piacentini, 1998). It is presumed that activation of TGase 2 during apoptosis prevents release of soluble and immunogenic proteins from dying cells because protein crosslinking makes these proteins less soluble, and thereby decreases a possibility of induction...

Simultaneous Measurement Of H2o2 And O2 In Neutrophils

This protocol combines the above two protocols in a single assay. This technique has the advantage of differentiating between H2O2 and O2- but still provides quantitatively valuable information from the assay system. Because DCF and EB have quite different emission frequencies, there is very little fluorescence overlap. Additional Materials (also see Basic Protocol) 10 mM hydroethidine (HE, Polysciences MW 315) in DMSO (prepare fresh and keep on ice, protected from light) 1. Prepare cell...

Combination Dnarna Fish and Immunophenotyping

Fluorescence-based detection methods allow the simultaneous detection of different DNA or RNA target sequences, together with proteins or other cellular constituents, in the same cell. Using combinations of fluorochromes having different excitation and emission spectra, these different cellular components can be identified on the basis of the different colors assigned to them. This approach is used in so-called genotype phenotype analysis to identify chromosomal aberrations in subpopulations of...

Design

Confocal microscopy is not a new technology. It was invented in 1955 by Marvin Minsky, a Harvard postdoctoral fellow who was trying to see the interconnections between neuronal cells (Minsky, 1957 1988). The large number of stained cells present in the sample and the large amount of light scattered by them into the field of view blurred the image and made it impossible to resolve individual cells with a conventional light microscope. His device, patented in 1957, is illustrated in Figure 2.8.1....

Commentary

Scanning laser cytometry of tissue imprints enables the study of functional aspects of solid tumors at the protein level (Kamentsky and Kamentsky, 1991 Gorczyca et al., 1997). Because such information is becoming increasingly important in the clinical management of breast cancer, the methods presented here focus on this tumor type. DNA aneuploidy and high tumor growth fractions are independent, unfavorable prognostic markers in breast cancer (Bergers et al., 1997 Fitzgibbons et al., 2000). The...

Prepare cells

Centrifuge white blood cell suspension 5 min at 350 x g, 20 C, and resuspend in S0rnes's buffer to a final concentration of 5 x 106 leukocytes ml. 2. For reference sample, add 2 ml DPBS containing 0.2 EDTA, 100 l cell suspension, and 500 l S0rnes's buffer to each of two 12 x 75-mm tubes. These tubes contain the same volume as the final dilution in the lower well, and are the 100 cell count. 3. Add 600 l S0rnes' s buffer to the lower chamber of two wells of the 24-well transwell microtiter plate...

Materials

Tissue sample with cells to be stained (e.g., resected tumor) Citrate DMSO buffer (see recipe) Internal DNA content standard (e.g., chicken or trout erythrocytes see recipe) Cell lysis solution with trypsin (see recipe) Trypsin-inactivating solution (see recipe), ice cold Propidium iodide (PI) spermine staining solution (see recipe), ice cold 0.5 x 25-mm needles (25 G x 1 in.) 10-ml disposable syringes 38 x 12.5-mm polypropylene tubes with caps 24- to 34- m nylon mesh Flow cytometer with 488-nm...

Basic Protocol 1

Holmes, Larry M. Lantz, and Wesley Russ Current Protocols in Cytometry (1997) 4.2.1-4.2.12 Copyright 1997 by John Wiley & Sons, Inc. 1. Dialyze purified monoclonal antibody against 500 ml FITC labeling buffer at 4 C with two or three changes over 2 days. Allow > 4 hr between buffer changes. This step removes free NH4+ ions and raises the pH to 9.2. Generally, up to 5 ml antibody can be dialyzed against 500 ml buffer. For discussion of dialysis and a detailed...

Bivariate Analysis Of Dna Content And Cyclins

This protocol combines measurement of DNA content with expression of either one of the D-type cyclins or cyclins E, A, or B1. Cells are fixed and labeled with cyclin antibody followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody, and finally stained with propidium iodide (PI) for DNA measurement. If directly conjugated FITC-anti-cyclin antibody is available, secondary antibody labeling (steps 6 and 7) can be omitted. This analysis is adapted to the most commonly used...

Hypogammaglobulinemic Serum

Convalescent serum is used as a source of specific antibodies. Acute-phase Healthy human serum Hypogammaglobulinemic serum Acute-phase and convalescent serum S0rnes's buffer (see recipe) For a pooled serum from healthy controls 1a. Collect serum (see Support Protocol 2) from healthy individuals, and ensure that CH50 is within the normal reference range. 2a. Mix equal volumes of serum from each of seven healthy individuals and label as pooled human serum (PHS). Heat-inactivate a...

Otto II buffer

Filter through a 0.22- m filter store up to 6 months at room temperature. Prior to use (if stain is to be incorporated directly in the buffer), add 0.1 mg ml DAPI stock solution (see recipe) to a final concentration of 4 g ml or 1 mg ml PI stock solution to a final concentration of 50 g ml along with 1 mg ml RNase stock solution (see recipe) to a final concentration of 50 g ml. RNase is needed to prevent PI from binding to RNA. Since DAPI specificially stains DNA...

Image Processing Software

There are a number of image processing and data analysis software packages available (see the November 1995 issue of IEEE Spectrum volume 32, no. 11 for reviews). These can be used for developing and testing algorithms and for routine processing in biomedical research work. Some of the more popular ones are listed, roughly in order of increasing cost, in Table 10.5.1. In most cases demo versions are available at no charge. Because the various packages differ in emphasis and user interface...

Univariate Flow Karyotyping And Chromosome Sorting Of Plant Chromosomes

This protocol describes the analysis and sorting of plant chromosomes stained with DAPI. The flow cytometer must be equipped with a UV light source to excite this dye. Chromosome suspension (see Basic Protocol 2) 0.1 mg ml DAPI stock solution (see recipe) LB01 lysis buffer (see recipe) Collection liquid Sheath fluid SF50 for flow cytometric analysis 40 mM KCl 10 mM NaCl (sterilize by autoclaving) Computer with spreadsheet or other software for theoretical flow karyotypes (available from...

Modern Objectives

Achromats, Fluorites, and Apochromats Achromats are corrected to bring at least two colors of light, usually red and blue, to a common focus and are most highly corrected for spherical aberration in the apple-green portion of the spectrum. Thus, when a green filter is used in the illumination path, modern achro-mats will yield quite good images. Recently designed achromats may have chromatic correction for three wavelengths and spherical correction for two. They are well suited for use in the...

Direct Versus Indirect Methods

In direct in situ hybridization, the fluorescent reporter molecule is bound to the nucleic acid probe so that hybrids that have formed can be visualized microscopically immediately after in situ hybridization (Wiegant et al., 1991). In indirect procedures, the probe contains an element that renders it detectable by additional labeling steps (e.g., biotin-streptavidin binding or immunocytochemistry) hence the term indirect. A number of such hapten modifications have been described. Direct...

Autofluorescence

Autofluorescence throws a small kink into compensation, but, as it turns out, does not change one's ability to deconvolute spillovers. Cellular autofluorescence is present in all detectors to varying extents, and provides a background that varies from cell to cell. There are three ways to deal with autofluorescence. One way is to devote a single detector to autofluorescence measurement. Because the autofluorescence spectrum of similar cells is generally identical, autofluorescence can be...

Preparation of Coated Slides

As a result of the proteolysis step, isolated cells or parts of tissue sections may be lost during the subsequent denaturation and hybridization steps, especially when noncoated glass slides are used. This can be avoided by coating the slides using either poly-L-lysine or aminoalkyl silane (organosilane). For proper adhesion of biological material, slides are electrostatically charged to attract single cells or frozen, formalin-fixed tissue sections. Coated slides can be prepared by dipping in...

Multidimensional Gating To Separate Overlapping Populations

Multidimensional data analysis can be used to separate populations seemingly inseparable in any two-dimensional plot. Figure 10.4.3 shows four-parameter data that were computergenerated using WinList 2.0 (Verity Software) to mimic immunofluorescence data. Two populations were created with centers at channels 16 and 28 (128 channels full scale) in each of the four parameters. The two populations had the same dispersion around the central points with respect to each of the four parameters. Six...

Care and Maintenance of Optical Filters

Colored glass filters are relatively robust but should be kept free of fingerprints while in use or storage. Clean with lens tissue as you would eyeglasses and handle around edges. Final cleaning with alcohol on a Q-tip cotton swab followed by wiping with lens tissue will remove most fluorescent materials that might be present. Colored glass filters should be very stable unless used directly in high-intensity light. These filters should be inspected visually for unevenness in color about every...

Image Processing and 2D Morphometry

The relative ease of processing and extracting information from digital images has led to a rapid growth in the use of image processing analysis and morphometry (units 10.5& 10.10). As defined by Weibel (1979), morphometry is the quantitative description of a structure. Currently, there are two different approaches to extracting measurements from images. The model-based method commonly employed in image-analysis software programs uses algorithms to extract measurements (e.g., area, perimeter...

Critical Parameters and Troubleshooting

A clean blood draw and gentle handling of specimens are required to avoid spontaneous platelet activation. If agonists are being added during processing to study cell reactivity, platelet aggregation could occur and interfere with analysis. It is important, therefore, that the samples be left undisturbed during any activation steps, followed immediately by fixation. Even in the absence of added agonist, LPA will form relatively quickly once blood is drawn into anticoagulant therefore, the time...

Overview of Nucleic Acid Analysis

A variety of fluorochromes can be used for flow cytometric analysis of nucleic acids. Their spectral properties, chemical structures, and binding characteristics are presented in detail in unit 4.2. This overview, therefore, is limited to general introduction of the methods presented in units 7.2-7.5, and primarily focuses on the applicability of these methods in different fields of biology and medicine. Analysis of nucleic acids is, perhaps after immunophenotyping (units 6.2 & 10.4), the...

DNA Content Measurement for DNA Ploidy unit 75 and Cell Cycle Analysis

In flow cytometry, analysis of DNA ploidy (DNA index or DI) and or discrimination of cells in G0 G1 versus S versus G2 M phases of the cell cycle is generally done by measuring cellular DNA content alone. Indeed, univariate DNA content analysis is an established clinical assay in oncology and is also widely used for research in cell and molecular biology (see unit 7.1 for an overview of nucleic acid analysis). A large number of DNA fluorochromes can be used for this purpose, and the binding...

PH Measurements With Snarf1 Using Nigericin Calibration

For most purposes, SNARF-1 is the most satisfactory pH probe currently available. It has a clear isobestic point i.e., a fluorescence emission wavelength that is insensitive to pH change , and it is very sensitive to pH changes within the physiological range. Calibration is achieved by resuspending dye-loaded cells in high-potassium buffers in the presence of the proton ionophore nigericin. Nigericin allows exchange of H for K across their concentration gradients, so that when external and...

Other Microscopy Techniques

Just as the rising or setting sun will better reveal the topography and mountain ridges of a landscape than the noonday sun, obliquely illuminating a specimen with limited internal contrast can greatly enhance structural differences in optical density or refractive index and turn an otherwise flat or almost invisible object into an image of striking relief and apparent three-dimensionality with clearly enhanced contrast Fig. 2.1.3 . To obtain oblique illumination with some degree of...

Principles of Quality Control

The following is a general overview of the principles of quality control in the clinical laboratory arena. Please refer to units 1.1 amp 1.3 for definitions of terms and flow cytometry-spe-cific designations. Here, the reader will become aware of historical clinical laboratory procedures and how they apply to cytometry. A basic premise of quality control is that the reported laboratory values should correspond to the correct or expected values. To examine this in more analytical terms, let us...