Measurement of apoptosis of intact human islets by confocal optical sectioning and stereologic analysis of YO-PRO-1-stained islets.
Apoptosis is an established pathway for islet cell demise. Current protocols for assessment of islet cell apoptosis are time-consuming (as with terminal deoxynucleotide transferase-mediated dUTP nick-end labeling reaction) and involve disruption of the islet architecture (as with flow cytometry) or destruction of cell integrity (as with enzyme-linked immunosorbent assay). The membranes of apoptotic cells, but not those of live cells, are permeant to the DNA-intercalant dye YO-PRO-1. We report a novel methodology for the rapid quantification of apoptosis of human islets: confocal laser optical sectioning and stereologic analysis of intact human islets stained with YO-PRO-1 and Hoechst 33342. The advantages include (1) rapid quantification of apoptosis without disrupting islet architecture and (2) identification of significant heterogeneity in the extent of apoptosis among islets from the same isolate. Confocal laser scanning microscopy microscopic imaging of YO-PRO-1-stained islets may advance investigation of islet cell apoptosis and help develop islet parameters predictive of posttransplant function.
Boffa
Daniel J
DJ
Department of Surgery, The New York Presbyterian Hospital, Weill-Cornell Medical Center, New York, NY 10021, USA.
Waka
John
J
Thomas
Dolca
D
Suh
Sungwook
S
Curran
Kevin
K
Sharma
Vijay K
VK
Besada
Melissa
M
Muthukumar
Thangamani
T
Yang
Hua
H
Suthanthiran
Manikkam
M
Manova
Katia
K
D017209:Apoptosis; D001583:Benzoxazoles; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology*; D018613:Microscopy, Confocal; D011806:Quinolinium Compounds; D013194:Staining and Labeling
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C089813:YO-PRO 1