There are some technical issues concerning the accurate flowcytometric Immunophenotyping(It is essential to take several factors into consideration in order to conduct accurate flowcytometric immunophenotyping.). We evaluated following factors: (1) the fluorescence intensity of major fluoroscence probes used for monoclonal antibody conjugation.;(2) the compensation requirements for the third color dye, such as PerCP, PECy5, PerCP-Cy5.5 ;(3) the reactivity differences resulted from clones used and sample treatments (ie., fixation). Results demonstrated that (1) antibodies labeled with bright fluorescence should be used to detect markers expressed at low levels on cell surface when determined with two-color combination for CD4 and CD25;(2) in 4-color analysis using APC conjugated antibodies, substantial compensation adjustment was required when used a tandem dye as the third color, particularly PE-Cy5, as a result of direct excitation by a 2nd red laser; (3) antibody reactivity was dependent on clones used and methods of sample preparation represented by CCR5 antibody.
