Pulse profile analyses of endocytosis in capped B lymphocytes and BCL₁ cells

Abstract

The effect of temperature on the kinetics of endocytosis by B lymphocytes and BCLl cells was examined by using flow cytometry. Mouse B cells were stained with FITC-RaMIg and induced to undergo cap formation in the temperature-controlled sample compartment of the flow cytometer. Capping and subsequent endocytosis were measured by continually monitoring the pulse profile descriptors (width and area) of the electronic signal curves generated during flow cytometric analyses. Decreases in area values which immediately followed cap formation were shown to result from a pH-dependent quenching of fluorescence emission as the internalized FITC-RaMIg entered acidic subcellular compartments. Similar results were obtained with BCLl cells, but, in addition to cap formation and acidification, cytoplasmic diffusion of the fluorescent ligand could also be discerned by flow cytometry. With either cell type the rates of cap formation and endocytosis were shown to be temperature dependent with temperature coefficient (&lo) values of 2.0-2.7. Based upon Arrhenius plots of width and area changes, activation energies for capping and endocytosis ranged from -12-18 kcallmol. Key terms: Flow cytometry, fluorescence, immunoglobulinFlow cytometry has been used to follow endocytosis in various cell types. In some studies the susceptibility of a ligand to labeling with either fluorescent antibodies (4,10,11) or liposomes (20) was used to follow internalization. However, in other studies workers capitalized on the pH sensitivity of fluorescein fluorescence emission to assay entry of fluorescein-conjugated ligands into acidic intracellular compartments (5,(12)(13)(14)(15)(16)18,21). Because similar studies might provide additional information regarding the endocytic pathway functioning in B lymphocytes and in leukemic B cells which have been employed in antigen processing and presentation studies (2,7), we have used flow cytometry to assess the kinetics of endocytosis following cap formation in B cells and in the BCLl cell line. For these studies we have made use of a previously described flow cytometric technique which involves the continuous monitoring of pulse area and pulse width values of the electronic signal curves generated by passage of the cells through the laser beam of the flow cytometer (3). As a result of these analyses, we were able to determine the rates of four consecutive steps in the endocytic pathway: capping, internalization, intracellular movement and entry of the fluorescent ligand into acidic subcellular compartments. In addition, by assaying the cells at various temperatures, we have been able to calculate Qlo and activation energy values for the four sequential steps. MATERIALS AND METHODSAnimals BALBk mice were obtained from a colony maintained in the Department of Microbiology at the University of Mississippi Medical Center. Isolation of B LymphocytesThe procedures used to obtain highly purified B lymphocytes have been described previously (1,3,23). Briefly, BALBic mice (2-3 months old) were killed by cervical disl...

Pulse Width for Particle Sizing

“…Rudolph et al used forward light scatter pulse width to estimate cell surface area and calculate the surface density of transferrin receptors (Rudolph et al, 1985). Yang et al used fluorescence pulse width to study the capping and endocytosis of fluorescently stained IgG on mouse B cells (Yang et al, 1988).…”

Cellular Pathway(S) of Antigen Processing and Presentation in Fish APC: Endosomal Involvement and Cell‐Free Antigen Presentation

“…Catfish Monocyte Lines. As in previous studies (Vallejo et al, 1991a(Vallejo et al, , 1991b (Cuchens and Buttke, 1984;Yang et al, 1988 (Ohkuma and Poole, 1978;Yang et al, 1988). Lysosomotropic agents like chloroquine, which raise intracellular pH (Ohkuma and Poole, 1978;Ziegler and Unanue, 1982;Seglen, 1983;McCoy, 1990), usually permit recovery of the signal.…”