Forward scatter pulse width signals resolve multiple populations of endosomes
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...
Saved in:
Published in | Cytometry (New York, N.Y.) Vol. 14; no. 4; pp. 411 - 420 |
---|---|
Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Hoboken
Wiley Subscription Services, Inc., A Wiley Company
1993
Wiley-Liss |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | 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. |
---|---|
Bibliography: | This work was supported by grants from the National Kidney Foundation of Wisconsin, the Wisconsin Affiliate of the American Heart Association, and Comprehensive Cancer Center grant P30CA14520 from the National Cancer Institute. The study was completed during Dr. Hammond's tenure as an American College Of Physicians Teaching and Research Scholar. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0196-4763 1097-0320 |
DOI: | 10.1002/cyto.990140410 |