PBMC from RA patients before and after treatment were incubated with
recombinant human IL-2 (40ng/ml; Gibco) for 3 hours in 12-well plates at 5
× 105 cells/well. Then, B cells were purified as described
above. To test whether CD39 on B cells is a functional E-NTPDase, purified B
cells from RA patients, obtained before or after 3 months of treatment, were
seeded into a 96-well plate at a density of 50000 cells per well in DMEM without
phenol red (Gibco #21063). The hydrolysis of ATP in the culture medium by CD39
was assessed by a bioluminescent assay. Cells were incubated with 10μM
ATP (Sigma-Aldrich A6419) in culture medium without serum, and the changes in
eATP concentrations, after 40 minutes at 37°C and 5% CO2, were
measured with an ATP bioluminescent assay kit (Invitrogen A22066) following
manufacturer’s instructions. Cell-free medium with ATP alone was used as
control. Briefly, 10 ul of culture supernatant was incubated with 90 ul of the
reaction buffer in a 96-well plate at 28°C. After 15 minutes, the
bioluminescence was measured at 560nm using the BIO TEK microplate
spectrophotometer (Bio Tek instruments, Inc.). The values obtained of each
experimental condition were referred to the value from medium culture alone with
ATP, which is considered 100% of ATP.
recombinant human IL-2 (40ng/ml; Gibco) for 3 hours in 12-well plates at 5
× 105 cells/well. Then, B cells were purified as described
above. To test whether CD39 on B cells is a functional E-NTPDase, purified B
cells from RA patients, obtained before or after 3 months of treatment, were
seeded into a 96-well plate at a density of 50000 cells per well in DMEM without
phenol red (Gibco #21063). The hydrolysis of ATP in the culture medium by CD39
was assessed by a bioluminescent assay. Cells were incubated with 10μM
ATP (Sigma-Aldrich A6419) in culture medium without serum, and the changes in
eATP concentrations, after 40 minutes at 37°C and 5% CO2, were
measured with an ATP bioluminescent assay kit (Invitrogen A22066) following
manufacturer’s instructions. Cell-free medium with ATP alone was used as
control. Briefly, 10 ul of culture supernatant was incubated with 90 ul of the
reaction buffer in a 96-well plate at 28°C. After 15 minutes, the
bioluminescence was measured at 560nm using the BIO TEK microplate
spectrophotometer (Bio Tek instruments, Inc.). The values obtained of each
experimental condition were referred to the value from medium culture alone with
ATP, which is considered 100% of ATP.