TY - JOUR
T1 - Quantitative measurement of Fc receptor activity on human peripheral blood monocytes and the monocyte-like cell line, U937, by laser flow cytometry.
AU - Christensen, Jesper Aagaard
AU - Leslie, R G
N1 - Keywords: Antigens, Differentiation; Binding, Competitive; Cell Line; Flow Cytometry; Humans; Immunoglobulin G; Monocytes; Receptors, Fc; Receptors, IgG
PY - 1990
Y1 - 1990
N2 - The expression of high affinity Fc receptors for IgG (FcRI) on the cell line U937 has been measured by flow cytometry, using fluorescein isothiocyanate-conjugated human IgG (FITC-IgG) calibrated spectrofluorimetrically against lasergrade fluorescein (Fl). A standard curve is presented, relating the effective fluorescence in terms of fluorescein equivalents per IgG molecule, to the degree of conjugation of FITC-IgG. The flow cytometer was calibrated with commercially available fluorescein-coupled latex beads. The quantitation of FcRI, in terms of sites per cell and affinity constants, was compared with a radioligand assay performed concurrently on the same cell population. Good agreement between the two assays was observed. The Fc receptors on peripheral blood monocytes were measured in unpurified lysed blood by gating on forward/side scatter. Monomer IgG binding to monocyte FcRII or FcRIII cannot be measured in direct IgG radioligand analyses because of the low affinity of these receptors and their low numbers per cell. However, flow cytometry may be employed for measuring both high and low affinity ligand-FcR interactions, using monomer FITC-IgG.
AB - The expression of high affinity Fc receptors for IgG (FcRI) on the cell line U937 has been measured by flow cytometry, using fluorescein isothiocyanate-conjugated human IgG (FITC-IgG) calibrated spectrofluorimetrically against lasergrade fluorescein (Fl). A standard curve is presented, relating the effective fluorescence in terms of fluorescein equivalents per IgG molecule, to the degree of conjugation of FITC-IgG. The flow cytometer was calibrated with commercially available fluorescein-coupled latex beads. The quantitation of FcRI, in terms of sites per cell and affinity constants, was compared with a radioligand assay performed concurrently on the same cell population. Good agreement between the two assays was observed. The Fc receptors on peripheral blood monocytes were measured in unpurified lysed blood by gating on forward/side scatter. Monomer IgG binding to monocyte FcRII or FcRIII cannot be measured in direct IgG radioligand analyses because of the low affinity of these receptors and their low numbers per cell. However, flow cytometry may be employed for measuring both high and low affinity ligand-FcR interactions, using monomer FITC-IgG.
M3 - Journal article
C2 - 2145369
SN - 0022-1759
VL - 132
SP - 211
EP - 219
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 2
ER -