Forward scatter pulse width signals resolve multiple populations of endosomes

• Published in: Cytometry (New York, N.Y.) Vol. 14; no. 4; pp. 411 - 420
• Main Authors: Hammond, Timothy G., Majewski, Rebecca R., Morré, D. James, Schell, Kathleen, Morrissey, Lawrence W.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 1993; Wiley-Liss
• Subjects: Animals; Biological and medical sciences; Bufo marinus; Cell physiology; Data Display; Endocytosis; flow cytometry; Flow Cytometry - methods; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; kidney; Kidney Cortex - ultrastructure; Male; Microscopy, Electron; Molecular and cellular biology; offset; Organelles - ultrastructure; Particle Size; pulse processing; Rats; Rats, Sprague-Dawley; Scattering, Radiation; toad bladder; Urinary Bladder - ultrastructure; vacuolar ATPase; Flow cytometry; Intracellular transport; Vertebrata; Endocytosis; Mammalia; Rat; Rodentia; Quantitative analysis
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/cyto.990140410

The technique of pulse width analysis, developed to optimize cell size resolution in cell cycle kinetics, has not previously been applied to small particles such as endosomes. Offset is used to subtract a portion of the beam diameter from forward scatter pulse width signals to optimize visualization and discrimination of small particles. We identify multiple endosomal populations by offset pulse width of light scatter parameters. Specifically, linear forward scatter pulse width measurements reveal at least two populations of endosomes in the rat renal cortex, the rat renal papilla, and the luminal endothelium of the toad urinary bladder. Logarithmically amplified forward scatter pulse width measurements display the full dynamic range of these signals, resolving additional populations not manifest with linear amplification. To confirm that the endosomes observed were resolved from optical and electronic noise, we examined physiological function. The endosomes acidified after supplying ATP to the intrinsic membrane H+‐ATPase present. Further, electron microscopy of sorted endosomal populations from the toad urinary bladder confirmed identity and homogeneity of the fraction. Flow cytometric analysis of endosomal populations by multiparametric techniques including pulse width analysis of structural parameters and pulse height analysis of fluorescence from entrapped fluorophores allows identification, isolation, and quantification of multiple endosomal populations. © 1993 Wiley‐Liss, Inc.