Detection of lymphocyte subsets using three-color/single-laser flow cytometry and the fluorescent dye Peridinin chlorophyll-a protein

Abstract

The fluorescent dye, Peridinin chlorophyll A protein (PerCP) derived from dinoflagellate organisms (Glenodinium sp.) can be excited by a 488 nm laser and emits light with a large Stokes shift and no major spectral overlap with commonly used chromophores such as fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE). PerCP was conjugated directly to various mouse monoclonal antibodies (mAb) specific for human leukocyte markers or to avidin for use with biotinylated-mAb, and used to perform three color single-laser flow cytometry. The efficacy of this method was demonstrated by analyzing the heterogeneity of thymus T lineage subsets and B lymphocyte subsets in blood. CD4-CD8-, CD4+CD8+ and CD4+CD8- or CD4-CD8+ subsets differ in their expression of cell-cell interaction markers including CD18, CD28, CD44 and Leu 8, and activation/subset markers CD45RO, CD45RA and CD26. Some CD5+ peripheral blood B cells, unlike CD5-B cells, expressed CD45RO or high levels of CD54 (ICAM-1) suggesting the CD5+ B cell population contains activated lymphocytes. The availability of such an accessible method for three color analysis will make it possible to do routine three color monitoring of immunologic diseases such as AIDS, and autoimmune or periodontal diseases.

U.S.-Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standardization and validation of laboratory procedures

“…If lysis is performed prior to staining, care needs to be taken that: (1) antigenicity is unaffected by the lysing solution, (2) the lysing solution is removed and the cells washed thoroughly so the kinetics of antibody binding are not affected, and (3) a lysing solution is used that does not contain a fixative (the use of a lysing solution containing a fixative will alter cell viability and yield questionable results for surface marker analysis). Regardless of whether lysis is performed prior to, or after, staining of the cells with monoclonal antibodies, selection of a particular lysing reagent should be based upon the ability of the reagent selectively to remove only the mature erythroid cells, with minimal effects on other cell types in the specimen.…”

“…These reagents are of two basic types: single fluorochromes, which excite at 488 nm and emit in the high red spectrum (Ͼ630 nm), or tandem conjugates, which are composed of two distinctly different fluorochromes (85). The primary example of a single dye is peridinin chlorophyll-A protein (PerCP) (1,54). This fluorochrome is quite ''user friendly'' in that it requires little color compensation correction for use with FITC and PE and exhibits little, if any nonspecific binding to myeloid cells.…”

Coexpression of CD69 and HLADR activation markers on synovial fluid T lymphocytes of patients affected by rheumatoid arthritis: a three‐colour cytometric analysis.

“…Monoclonal antibodies and flow cytometric analysis. SF and PB mononuclear cells were stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled anti-CD69, anti-HLADR, anti-CD25 monoclonal antibodies (mAbs) (Becton Dickinson), in combination with peridinin chlorophyll protein (PerCP) conjugated anti-CD3 antibody (Becton Dickinson) (Afar et al 1991). Isotype-matched mouse mAbs not reactive with human leucocyte were used as control.…”

Evidence for the continuous recruitment and activation of T cells into the joints of patients with rheumatoid arthritis

“…Three-color immunofluorescence was performed as previously described [21]. Briefly, cell suspensions, pre-incubated with 2% normal human serum, were incubated for 20 min on ice with mAb directly conjugated to peridinin chlorophyll protein (PerCP), phycoerythrin (PE) or fluorescein isothiocyanate (FITC).…”