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using
the
Ficoll-Hypaque
technique
(Amersham
Biosciences;
Buckinghamshire, UK). The samples were checked for purity by FACS, and the
procedure was repeated if the percentage of cells other than CD19 + B cells exceeded
5%. Non-manipulated peripheral blood B cells were isolated from the PBMCs of healthy
donors by negative selection using the RosetteSep isolation kit for B cells (STEMCELL
Technologies; Vancouver, Canada). Cell type (CD19 +) purity was at least 95%. In some
experiments (n=3), the separation method of sheep red blood cells was used to obtain
distinct populations of B and T cells from the same B-CLL patients.
peroxidase
or
alkaline
phosphatase
and
were
detected
by
Cell treatment
CTLL-2 cells were treated as previously reported 6. Next, 1 106 B and T cells
either from patients or from healthy subjects were incubated with the various
compounds (as indicated in figure legends) for 24 hs in a 96-well plate in 200 L
DMEM without serum and phenol red. Psora-4 and PAP-1 were used at 20 M unless
otherwise indicated, clofazimine at either 1 or 10 M, as indicated. CSH was used at 4
M, and probenecid at 50 M.
targeted 0.5 M Q-7BTPI13 or SOD-PEG (60 U/mL) + CAT-PEG (300 U/mL; both from
Sigma) for 30 min at room temperature. In some panels the results are presented as
plots of the median of the fluorescence distribution histograms recorded under the
various conditions and incubation times, normalized by setting the values measured at
time zero as 100%.
Electrophysiological experiments
Whole-cell currents were recorded with an EPC-7 amplifier (List, Darmstadt,
Germany; filter, 1 kHz; sampling rate, 5 kHz) in B cells obtained as described above
and cultured in RPMI for a maximum of 24 hr, as described previously 8. Leak currents
were not subtracted. For whole-cell experiments the bath solution was composed of
150 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2, 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The
intracellular solution contained 134 mM KCl, 1 mM CaCl 2, 2 mM MgCl2, 10 mM EGTA,
and 10 mM HEPES (pH 7.3). Intracellular voltages are reported, and outward currents
are plotted upward.