Dye Saturation

With fluorescence, there is a limit to the sensitivity one can achieve by increasing excitation intensity or decreasing flow velocity. This limitation is due to the photochemical degradation of the dye, called bleaching. For example, a FITC molecule passing with a velocity of 4 m sec through a 488-nm Gaussian laser focus having a power of 1 W and a width of 100 m will absorb 140 photons. Every time the molecule is excited there is a certain probability (quantum yield) that it will dissociate or...

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...

Cds Fitc

Figure 6.8.9 Universal Template on a FACSCalibur. Analysis with a 4-color panel in one tube. The upper panel describes the absolute count with bright CD45 gating (Region A). The lower panel illustrates the percentage lymphocyte number with T-gate (Region B). Dot plot no. 5 CD3 versus CD4, ungated histogram no. 5 CD3, ungated 4. To define a fluorosphere region to permit the calculation of absolute cell numbers, create a fluorosphere region (R4 E) on dot plot no. 5 or set cursor (M1 E) on...

Cd45apc

Figure 10.15.6 Software compensation. The software has automatically compensated the uncompensated data shown in Figure 10.15.5. Parameters without any antibody of that color are designated by the parameter name. Otherwise, the antibody name and fluorochrome are used. The first row shows compensated CD45-FITC in FL1, the second row shows CD4-PE in FL2, the third row shows CD2-PE-Cy5 in FL3, and the fourth row shows CD45-APC in FL4. Regions R2 to R13 are drawn around each cluster. Daily...

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...

Antituberculosis agents

Use the following formula to prepare stock solutions of the following three antimy-cobacterial agents, based on the assay potency ( g mg) listed on each vial. All three agents can be obtained from commercial sources such as Sigma, the U.S. Pharmacopoeia Convention, and individual manufacturers. Store according to instructions included by the distributor or manufacturer. volume (ml) needed x agent concentration ( g ml) needed Ethambutol Dissolve in deionized distilled water to a concentration of...

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...

Flow Cytometric Assessment of HLA Alloantibodies

Antibodies against HLA molecules are formed in response to exposure to foreign HLA molecules, which can occur as a result of blood transfusion, pregnancy, or transplant. Blood components, particularly those containing cellular elements, are the most common cause of anti-HLA antibodies. For a number of reasons, the accurate and sensitive detection of HLA antibodies is of critical clinical importance. The detection of donor-directed HLA antibodies in the serum of potential transplant recipients...

Digoxigeninlabeled Hybridization Probesprotocol

An alternate fluorescence protocol (see Basic Protocol 3) is presented for use with digoxigenin-labeled ISH probes. For lower sensitivity, the digoxigenin-labeled probe is detected by incubation with a fluorochrome-conjugated anti-digoxigenin antibody. As in Basic Protocols 1, 2, and 3, higher sensitivity is obtained by incubation with multiple antibodies. As with the first alternative antibody series for enzyme-based detection (see Tables 8.4.1 and 8.4.2), both secondary and tertiary...

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...