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...

Enzymatic Synthesis Of Padlock Probes

Enzymatic synthesis of padlock probes is recommended when probes needed are longer, more densely labeled than is possible through chemical synthesis. Chemically synthesized padlock probes are typically 70 to 90 nucleotides (nt). The authors have found that a probe length of 140 nt produced maximal detection signals of a repeated sequence in metaphase chromosomes. Probes > 200 nt gave rise to a low signal and high background due to unspecific binding (Antson, 2000). It should be noted, though,...

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...

Protocol10

Specimen Handling, Storage, and Preparation Handling, Storage, and Preparation of Human Blood Cells bent columns and the magnetic bead separation techniques, can be used in either a positive- or a negative-selection mode. Again, the manner in which these techniques are used depends on the cell surface antigens and their corresponding antibodies, as well as any effect these may have on cell function. Numerous points must be considered in using preenriching or separation techniques prior to flow...

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...

Info

Have an increased DNA stainability (Evenson and Melamed, 1983 Engh et al., 1992 ), which can be visualized by univariate analysis (Engh et al., 1992) as well as by the SCSA bivariate analysis. In most cases, the defects of high DNA stainability and DNA denaturation are mutually exclusive, and any single cell rarely has both defects. However, the authors have observed a limited number of human clinic cases where both defects occur within the same sample. Figure 7.13.1 illustrates how to resolve...

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...

Background Information

Assessments of viability depend on one or both of two cellular properties 1 the intact-ness of the cell membrane, and 2 the physiological state of the cell. Dye exclusion methods are based on the fact that only intact membranes are impermeable to large or charged molecules. Intact membranes also maintain cytoplasmic gradients with respect to the surrounding medium, thus retaining intracellular concentrations of ions and small molecules. This latter property also reflects the physiological state...

Basic Protocol

Contributed by Sue Chow and David Hedley Current Protocols in Cytometry 1997 9.3.1-9.3.10 Copyright 1997 by John Wiley amp Sons, Inc. 2. For each milliliter of cell suspension, add 2.5 Ml of 2 mM carboxy SNARF-1 AM 5 MM final and incubate 30 min at 37 C. 3. Centrifuge at room temperature, selecting time and g force as appropriate for the cell line used. Resuspend dye-loaded cells in 10 Ml PBS for each milliliter of cell suspension stained 1 x 10s cells ml final . 4. Pipet 0.5 ml of each...

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...