Fluorochrome-labeled

Immunophenotype The process of identification and quantitation of cellular antigens through fluorochrome-labeled monoclonal antibodies. 

Hopman AHN, Ramaekers FCS, Speel EJM (1998) Rapid synthesis of biotin, digoxigenin, trinitrophenyl, and fluorochrome labeled tyramides and their application for in situ hybridiza tion using CARD amplification. J Histochem Cytochem 46 771 777 Ino H (2003) Antigen retrieval by heating en bloc for pre fixed frozen material. J Histochem Cytochem 51 995 1003... 

Isotype-matched unconjugated or fluorochrome-labeled antibodies unreactive with mammalian cells are available as control reagents from many suppliers. 

Fluorochrome-labeled anti-immunoglobulin antibody labeled F(ab )2 fragments should be selected for target cells that express Fc receptors... 

Dispense 100 pL of fluorochrome-labeled antibody solution (10 pg/mL) into tubes (see Note 3) Control fluorochrome-labeled antibody of the same isotype should be used m control tubes at the same concentration... 

Add 100 pL of fluorochrome-labeled anti-immunoglobulin reagent, diluted in wash medium (see Note 11). 

Having considered conditions for the binding of fluorochrome-labeled antibody to reflect antigen density, the most appropriate flow cytometric analysis method can then be selected. Some of these considerations will be specific to particular manufacturers flow cytometers and cannot be addressed in detail here there are, however, common approaches, that are generally applicable, and appreciation of the principles involved will at least allow some of the more obvious pitfalls to be avoided. 

Unbound fluorochrome-labeled antibody may need to be removed to allow the analysis of cell-bound labeled antibody. However, it should be noted that with... 

The dissociation rate constant of an antibody-antigen interaction is characteristic of individual antibodies, but as discussed above, the rate at which antibody falls off the cell also depends on the valency of binding. Monovalent dissociation rates are faster than divalent dissociation rates, and the reassociation of a divalent interaction (i.e., of an antibody already bound by one binding site) is favored in comparison with monovalent association from the fluid phase. This avidity effect means that divalently bound fluorochrome-labeled antibodies are shed more slowly when fluid-phase antibody is removed by washing. 

Robins, R A, Laxton, R R., Garnett, M., Price, M. R., and Baldwin, R W (1986) Quantitation of antitumor antibody and antibody conjugate binding activity by competition with fluorochrome-labelled antibody J Immunol Methods 90, 165—172. 

When one or more of the desired antibodies is not available as a direct conjugate and a conjugate cannot be synthesized, indirect methods have to be used. The simplest variation is to use a biotin-labeled antibody as a first layer, and then after washing the cells, incubate with fluorochrome-labeled streptavidin, and additional directly labeled antibodies labeled with complementary fluoro-chromes. The additional streptavidin step does not usually cause difficulty... 

Fig. 8.24. Cells in late apoptosis have fragmented DNA. They can be visualized as cells with increased incorporation of fluorochrome-labeled nucleotides to the ends of the fragments in the flow cytometric TUNEL assay (lower dot plots, with FL1 as FITC-dUTP and FL2 as propidium iodide bound to DNA). Alternatively, they can be visualized as cells with less-than-normal (sub-GO/Gl) DNA content (upper histograms of propidium iodide fluorescence). Data from etoposide-treated ML-1 cells courtesy of Mary Kay Brown and Alan Eastman.

Detach exponentially growing HeLa pLuc705 cells (3.5 x 105) with trypsin (0.05%)-EDTA-4Na (0.35 mAf), centrifuge at 900 x g for 5 min, resuspend in 2 mL OptiMEM, and incubate with the fluorochrome-labeled conjugates (between 1 and 2.5 pM) for 30-120 min. 

Figure 9.8 Evaluation of binding of biopharmaceutical X to cynomolgus monkey peripheral blood cells. Cynomolgus monkey cells were stained with fluorochrome-labeled biopharmaceutical X or the natural ligand for the human receptor. Expression of the target receptor on the cynomolgus cells was detected using antibodies specific for the target receptor.

Bedner, E., Smolewski, P., Amstad, P. A., and Darzynkiewicz, Z. A. (2000). Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA) Correlation with DNA fragmentation. Exp. Cell Res. 259, 308—313. 

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