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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...

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...

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...

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...

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...

Other Microscopy Techniques

Cardioid Microscope Condenser

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,...