Membrane Potential and Changes in Ion Permeability

The generation of intermediate oxygen radicals after monocyte and neutrophil stimulation is accompanied by changes in membrane potential. Lipophilic dyes such as the cyanine compounds dipentyloxacarbocyanine (DiOC5 3 ) and dipropyl-thiocarbocyanine (DiSC5 3j) can be used to measure this aspect of cellular activation. These dyes diffuse into the cell with different localization patterns depending on their concentration. Cellular activation is followed by a loss of Fig. 3. Flow cytometric...

References

G. (1993) A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood. J. Immunol. Methods 163, 155-160. 2. Macey, M. G., Jiang, X. P., Veys, P., McCarthy, D., and Newland, A. C. (1992) Expression of functional antigens on neutrophils. Effects of preparation. J. Immunol. Methods 149, 37-42. 3. Macey, M. G., McCarthy, D. A., and Newland, A. C. (1994) The ex vivo function and expression of function associated...

Priming and Activation

Priming refers to a process whereby the response of cells to an activating stimulus is potentiated, sometimes greatly, by prior exposure to a priming agent (Table 2). Neutrophil and monocyte priming by agents such as TNF-a, G-CSF, GM-CSF, and LPS causes a dramatic increase in the response of these cells to an activating agent. This process has been shown to be critical for phagocyte-mediated tissue damage both in vitro and in vivo. The principal consequence of priming (aside from direct effects...

The Choice of Negative Control

This is one of the most controversial issues in flow cytometry and one that has inspired many hours of discussion and exchange of e-mails. For decades, nascent flow cytometrists have been taught to include isotype-matched controls in all experiments to determine the background level of mAb binding and thus the threshold level for a positive event. This approach seems inherently sensible and attractive. However, the use of an isotype-matched control requires that one make many assumptions, most...

Calcium Flux Assay Procedure Using Fluo3

To measure the changes in intracellular calcium in cells in response to activating and control peptides, the calcium indicator Fluo-3 and flow cytometry may be used. Cells (2 X 106 mL) are incubated with Fluo-3 at a final concentration of 2 pM for 30 min at 37 C in Ca- and Mg-free Hanks' balanced salt solution (HBSS) buffered with HEPES (1 mM). Cells are then diluted 1 10 in buffered HBSS containing Ca and Mg. The baseline level of Fluo-3 fluorescence is determined in a dot plot of green...

Suppliers of Instruments Reagents Antibodies and Other Materials

Table 1 gives the internet addresses of some directories of suppliers which may be searched alphabetically by name or by product, whereas Table 2 gives the internet addresses of suppliers. Biotech Amgen Ltd Amrad (incorporates Systems Bangs Laboratories Inc. Beckman Coulter Biodesign International BioErgonomics instruments.co.uk http www.accurate-chemical.com http www.amgen.com http www.amrad.com http www.ancell.com http www.applied- cytometry.com http www.bagslab.com http www.beckman-...

Principles of Particle Sorting 31 Electrostatic Sorting

Most analytical flow cytometers are enclosed in that cells are aspirated from a reservoir and hydrodynamically focused so that they pass one by one through a light source, generally from one or more lasers. At this point, scattered light and fluorescence signals are generated, detected, and measured. After this, cells are removed under vacuum to a waste reservoir. In general, flow sorters use a principle involving the electrostatic deflection of charged droplets similar to that used in ink-jet...

Cell Preparation

For any flow cytometry application, cells must be prepared so that they are single and suspended in an appropriate medium. There are a variety of procedures for preparing cells, and the method used will depend upon the cell type being investigated. Once prepared, the cells may then be labeled with a fluorochrome-labeled mAb or fluorescent dye. The labeling procedure may be influenced by a number of factors, including the specificity of the antibody and the density of the antigen being...

Excluding Nonspecific Labeled Cells by Using a Dump Channel

To optimize the analysis, the best possible labeling strategy should be selected. A one-antibody strategy for identification of a certain population may be improved upon by using a second antibody that positively identifies the cells and by using a so-called dump channel. An example of the latter is shown in Figure 1. In Figure 1a, whole blood was labeled with an anti-blood dendritic cell antigen-2 BDCA-2 or CD303 monoclonal. This antigen is exclusively expressed by plasmacytoid DCs in human...

Labeling Procedure for Intracellular Cytokines

If a cell surface labeling is planned, this must always be carried out first, before fixation and membrane permeabilization. This part of the procedure should follow the standard protocol for the antibody or tetramer that is used. It is worth noting that because the cells will be fixed, it will not be possible to use dyes such as propidium iodide or 7-AAD for dead cell exclusion. It is thus important to use cells in good condition, and this becomes even more pertinent when considering that both...

Fluorescence Resonance Energy Transfer

If two different fluorochrome molecules are in close proximity, their electronic orbitals can interact by exciton or coupled oscillator mechanisms resonance . Consequently, energy absorbed by the one with the shorter excitation wavelength and therefore the higher energy absorption the donor can be passed to the other the acceptor , which fluoresces. The process is usually called fluorescence resonance energy transfer FRET but is also known as singlet-singlet or exciton resonance energy transfer...

The Measurement of Fluorescence Intensity

A regular use of flow cytometers is the determination of the density of specific molecules on the surface of one or more cells in a population. These measurements may be relative, semiquantitative, or quantitative depending upon the question asked and the reagents available. In most cases, the measurement of relative fluorescence intensity is adequate, where the fluorescent channel number that best approximates the average fluorescence of one population is compared with the same value from a...

Principles of Flow Cytometry

Flow Cytometry Principle

Flow cytometry is a powerful tool for interrogating the phenotype and characteristics of cells. It is based upon the light-scattering properties of the cells being analyzed and these include fluorescence emissions. This fluorescence may be associated with dyes or conjugated to mAbs specific for molecules either on the surface or in the intracellular components of the cell. Flow cytometry facilitates the identification of different cell types within a heterogeneous population. It was initially...