Wistar rats were treated with flutamide (s.c 7.5 mg/kg/day) or its vehicle for 5 days. Peritoneal cells were harvested after 4 h of thyoglycollate i.p injection, washed, and resuspended in HBSS as described above. Total cells were then plated onto 24 well plates (1 × 106 neutrophils/well in RPMI 1640) and pulsed ex vivo with LPS O55:B5 1 μg/ml for 24 h to elicit cytokine secretion. In vitro assays maintained the same conditions as in vivo (i.e., flutamide stimulation). Immunofluorescence for IL10 in neutrophils was carried out on methanol-fixed, permeabilized cells on coverslides using PE anti-rat IL10 (1/50; BD Biosciences, San Jose, CA) and FITC anti-rat Gr (1/50) antibodies.
For flow cytometry, cells were treated with cycloheximide 100 μM (Sigma Aldrich, St. Louis, MO) 3 h before the analysis. The cell concentration was adjusted to 5 × 105 cells/ml and labeled for 30 min with the following antibodies: PE anti-rat IL10 (1/60), APC anti-rat CD11b (1/100; BD Biosciences, San Jose, CA), and FITC anti-rat Gr (1/150). Signals were acquired in a FACSCanto II cytometer and analyzed using FlowJo X software (Tree Star, Ashland, OR).
For flow cytometry, cells were treated with cycloheximide 100 μM (Sigma Aldrich, St. Louis, MO) 3 h before the analysis. The cell concentration was adjusted to 5 × 105 cells/ml and labeled for 30 min with the following antibodies: PE anti-rat IL10 (1/60), APC anti-rat CD11b (1/100; BD Biosciences, San Jose, CA), and FITC anti-rat Gr (1/150). Signals were acquired in a FACSCanto II cytometer and analyzed using FlowJo X software (Tree Star, Ashland, OR).