Calibur

The cells were labeled with the fluorescent antibodies as described in
details elsewhere. [63 (link), 71 –72 (link),
79 ] They were sorted on the Calibur,
Vantage SE, or Aria (Becton-Dickinson). The antibodies were dissolved and all
washing steps carried in phenol-free, Ca+ / Mg+- free, PIPES buffered saline
solution, supplemented with 20 mM glucose, 10% human serum. Sorting was
performed on Aria, Calibur, Vantage SE (Becton-Dickinson) with the sheath
pressure set at 20 pounds per square inch and low count rate. The sorted batches
were analyzed on Calibur or Aria using FACS Diva software or on the FC500
(Beckman-Coulter). For the measurement of the fluorescently labeled cells, these
settings were tuned at the maximum emission for the Eu chelated antibody at 500V
with references to isotype antibodies and non-labeled cells. This assured the
comparisons between populations of cells labeled with multiple antibodies
without changing the settings on PMTs.
The fluorescently labeled cells or tissues were imaged with the Axiovert
(Zeiss) equipped with the Enterprise argon ion (457 nm, 488 nm, 529 nm lines)
and ultraviolet (UV) (364 nm line) lasers; Odyssey XL digital high-sensitivity
with instant deconvolution confocal laser scanning imaging system operated up to
240 frames/s (Noran), and the Diaphot (Nikon) equipped with the
diode-pumped Nd:YLF solid state laser (1048 nm line) (Microlase).

The cells were labeled with the fluorescent antibodies as described in
details elsewhere. [25 (link)–26 (link), 29 –30 ] They were
sorted on the Calibur, Vantage SE, or Aria (Becton-Dickinson). The antibodies
were dissolved and all washing steps carried in phenol-free, Ca+ / Mg+- free,
PIPES buffered saline solution, supplemented with 20 mM glucose, 10% human
serum. Sorting was performed on Aria, Calibur, Vantage SE (Becton-Dickinson)
with the sheath pressure set at 20 pounds per square inch and low count rate.
The sorted batches were analyzed on Calibur or Aria using FACS Diva software or
on the FC500 (Beckman-Coulter). For the measurement of the fluorescently labeled
cells, these settings were tuned at the maximum emission for the Eu chelated
antibody at 500V with references to isotype antibodies and non-labeled cells.
This assured the comparisons between populations of cells labeled with multiple
antibodies without changing the settings on PMTs.
The fluorescently labeled cells or tissues were imaged with the Axiovert
(Zeiss) equipped with the Enterprise argon ion (457 nm, 488 nm, 529 nm lines)
and ultraviolet (UV) (364 nm line) lasers; Odyssey XL digital high-sensitivity
with instant deconvolution confocal laser scanning imaging system operated up to
240 frames/s (Noran), and the Diaphot (Nikon) equipped with the
diode-pumped Nd:YLF solid state laser (1048 nm line) (Microlase).