Fluorochrome conjugated

Burns J, Hambridge M, Taylor CR. Intracellular immunoglobulins. A comparative study of three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. J. Clin. Pathol. 1974 27 548-557. 

View the specimen on a fluorescence microscope equipped with a filter combination appropriate for the fluorochrome conjugated to the secondary antibody (see Note 12). 

Some W-formyl peptide (FP) chemoattractants that are directly conjugated with fluorochrome (FP-Fl) are commercially available (Molecular Probes) and may be utilized in the assay. Alternatively, the peptide may be fluorochrome-conjugated and purified by the investigator. 

Fig. 6.6. The fluorescence histogram of a mixture of fluorochrome-conjugated calibration beads and the calibration line for channel numbers and their equivalence in soluble fluorescein molecules derived from that histogram. From Givan (2001).

Although many applications of flow cytometry involve the staining of cells for proteins expressed on the outer membrane, cells also have many proteins that are not displayed on their surface. With appropriate procedures, flow cytometry can provide a means to analyze these intracellular proteins. The outer cell membrane is impermeable to large molcules like antibodies however, if we intentionally fix cells to stabilize proteins and then disrupt the outer membrane, the cells can be stained with fluorochrome-conjugated monoclonal antibodies against intracellular proteins. After time to allow the antibodies to pass through the now-permeabilized membrane, the cells are washed to remove loosely bound antibodies and then are run through the flow cytometer to measure their fluorescence intensity. 

Receptor-specific antibodies and fluorochrome-conjugated reagents are diluted in PBS-gelatin or PBS-gelatin-Sap (for permeabilized cells) as required. Appropriate antibody dilutions should be determined empirically. Note Cells should not be allowed to dry at any stage in the procedure. 

Figure 9.3 Mean total lymphocyte count after one course of treatment. Peripheral blood was collected from individuals administered test article (circles) or placebo (squares) on a weekly basis, and subject to flow cytometry analysis to determine mean lymphocyte counts. A standard panel of fluorochrome-conjugated antibodies was used to identify the various lymphocyte sub populations. The solid bar indicates the dosing interval.

Cytotoxicity may decrease cell surface expression and transcription of markers. Thus, if concentration of test chemical induces low cell viability then measurement of cell surface expression and transcription is limited. In addition, donor variability may introduce differences in fluorochrome-conjugated monoclonal antibody expression (Aiba et al. 1997). 

The fluorochrome-conjugated monoclonal antibodies used may be varied. Investigation on one, a combination or all may be performed. In addition RNA Isolation to confirm the quality of RNA and Genochip Microarray for transcript profiling may be performed after flow cytometry evaluation. 

A variety of particles have been utilized in flow cytometric phagocytosis assays, including latex microspheres (7,8,12), bacteria ( 70,13,16), zymosan (9,13), and baker s yeast (11). Most of these particles are commercially available as fluorescent reagents. However, microorganisms may also be directly fluorochrome-conjugated (see Notes 1 and 2). 

Schuit et al. (1981) investigated the specificity of 29 fluorochrome-conjugated antisera against human IgG and found half of these commercial preparations unsatisfactory, indicating the frequent absence of adequate quality control. The adequacy of the antiserum should, therefore, be established by strict testing. 

Different combinations of fluorochrome-conjugated antibodies can be used depending on the available flow cytometer. 

Phosphatidylserine, which normally is on the inner leaflet of the plasma membrane, early during apoptosis becomes exposed on the outside cell surface (13). Because the anticoagulant protein annexin V binds with high affinity to phosphatidylserine, fluorochrome-conjugated annexin V can serve as a marker of apoptotic cells (21). During progression of apoptosis, the ability to bind annexin V precedes the loss of the plasma membrane s ability to exclude cationic dyes such as PI. 

Finally, the extremely low IgE concentration in the circulation requires very sensitive detection methods — i.e. ELISA/FEIA, RIA, or modifications of these methods. Basically, these techniques are similar to those described for the detection of autoantibodies. In the case of ELISA/FEIA, it concerns a capture technique where the IgE molecule is first captured by an anti-IgE monoclonal antibody and subsequently recognized by an enzyme/fluorochrome-conjugated anti-IgE monoclonal antibody. 

Key words Antibody, Immunolocalization, Fluorochrome, Conjugation,Thiol, Amine... 

Fluorescence-based immunolocalization of biomolecules within cells and tissues requires that fluorochromes, or fluorescent markers such as quantum dots, be covalently conjugated to antibodies. Techniques for antibody-fluorochrome conjugation were first devised by A. H. Coons and his associates, who pioneered the use of immunofluorescence microscopy in the 1940s and 1950s. Initially, the conjugation of fluorochrome to antibody was done directly to the antibody with the desired antigenic specificity (1). Subsequently, an indirect method (2) of immunolocalization was introduced that proved advantageous for most routine work. 

For indirect immunolabeling, the antibody of desired specificity, referred to as the primary antibody, is applied first, without fluorochrome conjugation. After excess primary antibody is washed away, a fluorochrome-conjugated, second antibody is introduced. Antigenic specificity of the second antibody is to immunoglobulin... 

For many researchers, the wide range of fluorochrome-conjugated secondary antibodies that are commercially available will likely obviate the need to undertake conjugation in the laboratory. Where it is necessary to do so, the researcher should give consideration to the commercially available kits (tee Note 6). Such kits provide a convenient way to obtain reliable and reproducible conjugation of fluorochromes antibodies or other proteins. 

Streptavidin-fluorochrome conjugates provide higher sensitivity than two-step indirect techniques. The use of Alexa dyes instead of the traditional fluorochromes enhances brightness and detection sensitivity of immunofluorescence methods several-fold (12). 

Remove third wash and add affinity-purified fluorochrome-conjugated secondary antibody, di-iuted as desired (typicaiiy, we use final concentration of 0.32-3.2 fig per ml) in MSM-Pipes in a final volume of 200 fi per well incubate 30 min at 37° in a humidified chamber. 

Incubate for 10 min with 100 /i.1 normal goat serum, followed by a 30-min incubation with 50 fluorochrome-conjugated secondary antibody in PBT. Wash two times for 3 min with 100 /i.1 PBT to remove excess antibody. If necessary, nuclei can be counterstained with a solution of 5 ju,g/ml DAPI (Sigma cat. D9542) in PBT for 5 min. 

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