Sperm Chromatin Structure Assay for Fertility Assessment

The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring. Damaged DNA in the single sperm that fertilizes a female oocyte can have a dramatic negative impact on fetal development and health of the offspring throughout adult life. Animal and human fertility clinics typically assess semen quality by measuring sperm density, total count, motility, and morphology. Clinics rarely measure sperm DNA integrity, perhaps because they are...

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...

Enumeration of Absolute Cell Counts Using unit 68 Immunophenotypic Techniques

At present the enumeration of absolute cell numbers is relevant in different research settings (e.g., cell culture) as well as in the clinical laboratory. Absolute counting of cells or cell subsets by flow cytometry is an established technique in at least three major clinical settings 1. Enumeration of residual leukocytes as part of the quality control of leukoreduced blood products 2. Enumeration of CD4+ and CD8+ T cells in HIV disease monitoring 3. Enumeration of CD34+ hematopoietic stem and...

Background Information

The role of platelets in the recruitment of monocytes and neutrophils to sites of vascular injury has long been recognized (Palabrica et al., 1992). The appearance of circulating LPAs is a dynamic process involving initial formation, vascular adhesion, potential sequestration to elements of the reticuloendothelial system, leukocyte activation, and LPA disaggregation via granulocyte proteinases (Gardiner et al., 2001). Circulating LPAs are increased in stable coronary artery disease (Furman et...

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...

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...

Specific Features Of Flow Cytometry Data Management

There are a number of areas in which improvements may be made in the management of flow cytometry data, including ways for users and flow cytometry facility staff to help maximize the usefulness and longevity of data. Most flow cytometry experiments are quite highly structured in ways that are not conveyed by simply listing the cells and reagents contained in each sample for example, some samples are controls other samples may constitute a time series or a reagent titration series or a number...

Immunophenotypic Analysis of Platelets

With an average diameter of 3 m, platelets are the smallest circulating cellular component in peripheral blood. The primary role of circulating platelets is to maintain hemo-stasis. The evaluation of platelets by flow cytometry has proven beneficial in the investigation of many disease states, including inherited defects such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, and storage pool disease (Michelson et al., 2001). Flow cytometric techniques have been used in blood bank...

Basic Protocol 2

Figure 11.10.1 DNA histogram for Saccharomyces cerevisiae n 70,000 after propidium iodide staining, indicating 41,000 G1 cells first peak and 22,000 G2 cells second peak . The third small peak consists of aggregated cells. The S-phase region lies between the two major peaks Hutter and Eipel, 1978 . Figure 11.10.1 DNA histogram for Saccharomyces cerevisiae n 70,000 after propidium iodide staining, indicating 41,000 G1 cells first peak and 22,000 G2 cells second peak . The third small peak...

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Figure 7.4.1 Measurement of DNA content combined with detection of apoptotic cells identified by the presence of DNA strand breaks, demonstrating the analysis of cell sensitivity to apoptosis in relation to the cell cycle phase. Apoptosis of HL-60 cells was induced by exposure to UV light UV , treatment with the DNA topoisomerase I inhibitor camptothecin CPT , y irradiation y RAD , or treatment with the DNA topoisomerase II inhibitor fostriecin FST as described Gorczyca et al., 1993b...

Labeling Antibody With Longarmed Biotin

Biotin is a naturally occurring vitamin with a molecular weight of 244 Da and an extremely strong affinity for avidin Kd 10 to 15 M-1 . Thus, biotin-labeled antibodies can be detected using commercially available avidin coupled to fluorochromes. Labeling antibodies with biotin provides flexibility by offering a choice of different fluorochromes to be used depending on the needs of the experiment. Moreover, because avidin has four binding sites for biotin and multiple biotin molecules can be...

Conjugation Of Texas Red To Rphycoerythrin To Produce An Energy Transfer Fluorochrome

An advantage of flow cytometric analysis is the ability to distinguish functionally significant populations of cells. The need to further subdivide these populations using antibody probes against known cell surface antigens requires increasingly complex multicolor analyses. Fluorochromes excitable with a single excitation source and possessing emission wavelengths distinct enough to be detected separately are needed to distinguish the different antigens. A highly effective way to achieve this...

Quantitative Determination Of Cell Surface Antigens By Direct Or Indirect Immunofluorescence Assay Using Quantum Simply

Quantum Simply Cellular Bangs Laboratories is a mixture of four uniform-size mi-crobead populations. Each bead population is labeled with a different calibrated amount of goat anti-mouse GAM IgG specific for the Fc portion of mouse IgG antibodies. The GAM is balanced against IgG1, IgG2a, and IgG2b isotypes and can be used as a universal calibrator for all mouse IgG monoclonal antibodies. The Quantum Simply Cellular beads are stained with the same fluorochrome-conjugated antibody and under the...

How To Measure Fret Efficiency

Energy Transfer Efficiency Distance

The energy transfer efficiency, as follows from the above formulas, can be determined in a number of different ways. Since energy is transferred from the excited donor to the acceptor, the lifetime t , quantum efficiency Q , and fluorescence intensity F of the donor decrease, if the acceptor is present Equation 1.12.10 . As a consequence, the fluorescence Figure 1.12.4 Distance dependence of the energy transfer efficiency. Distances are expressed in R0 units. The shaded area shows the useful...

Flow Cytometry Voltage Amp Gain

Figure 1.4.2 Proper settings for PMT anode voltage and amplifier gain. Panel A shows how the combination of low PMT anode voltage and high amplifier gain leads to a nonlinear response. Raising the PMT anode voltage and lowering the amplifier gain corrects the problem. Panel B shows how a low PMT anode voltage leads to a nonlinear response for large signals. Attenuating the light signal with an ND neutral density filter and increasing the PMT anode voltage produce a linear response. The...

Other Microscopy Techniques

Axial Illumination Photography

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...

Establishing System Linearity

Amplifier Flow Cytometry

The easiest way to establish the linearity of a flow cytometer is to analyze particles that fluoresce or scatter light with known relative intensities. Absolute intensities of the particles need not be known however, relative intensities must be known with an accuracy acceptable for the application for which the flow cytometer is being tested to run. The range of particle intensities is chosen to span the full histogram scale. By far the most common calibrators are fluorescent particles,...