The values of NS wells were subtracted from those of stimulated wells. The assay was

performed by strictly following the instructions of the BD™ ELISPOT Mouse IFN-γ ELISPOT Selleckchem HSP inhibitor Set (BD, San Diego, CA). Briefly, a 96-well ELISPOT plate was precoated overnight at 4 °C with anti-mouse IFN-γ capture antibody. After one wash with 200 μL per well of blocking solution, 200 μL of blocking solution was added to each well for 2 h at room temperature. The blocking solution was discarded, and a total volume of 100 μL of spleen lymphocyte suspension (adjusted to 2 × 106 cells mL−1) was added to each well. RPMI 1640 medium was supplemented with 10% v/v FBS. The cells were incubated in medium containing 2 μg mL−1 of PPD, 0.8 μg mL−1 of ConA, 16 μg mL−1 of Ag85b, 16 μg mL−1 of HspX, 16 μg mL−1 of C/E or medium alone (no stimulation). After incubation at 37 °C in 5% CO2 for 24 h, cells were removed, MG-132 mouse and the following steps were taken in strict accordance with manufacturer’s instructions. Spots were quantified using

an ELISPOT reader (Cellular Technology Ltd, Shaker Heights, OH). Four percent starch broth (1 mL) was injected into the peritoneum of mice 3 days before sacrifice to yield inflammatory macrophages. After sacrifice, mice were sprayed with 70% alcohol to sterilize the abdomen, then 2 mL of cold Hanks’ balanced salt solution without Ca2+ and Mg2+ (CMF-HBSS) was injected into the peritoneum. After slightly massaging the abdomen for several minutes, the fluid in the peritoneum was collected and centrifuged at 453 g for 10 min. Cells were washed twice with cold CMF-HBSS and then resuspended in Dulbecco’s minimum essential medium (DMEM) (Thermo Scientific) containing 5% FBS. Cells were stained with Diff-Quik staining solution for counting under a microscope. The cell concentration was adjusted to 2.5 × 106 cells mL−1. A 1-mL aliquot of the cell suspension was added to each well of a 24-well plate (Corning) and incubated at 37 °C in 5% CO2 for 2 h. Cells that did not adhere to the wells were discarded, and the wells were washed once with 37 °C DMEM. After the addition of 1 mL of 5% FBS-DMEM to

each well, stimulants were added at the following final concentrations: 10 μg mL−1 of Ag85b, 10 μg mL−1 of HspX and 10 μg mL−1 of LPS. Plates were Bcl-w incubated at 37 °C in 5% CO2 for 48 h, and then culture supernatants were harvested and stored at −80 °C until analysis. IL-12 was determined strictly following the instructions from the Quantikine Mouse IL-12 p70 kit (R&D Systems, Minneapolis, MN). Statistical analysis was performed using graphpad prism version 5.0 for Windows (GraphPad Software, San Diego, CA). Data analyses for antibody response, lymphocyte proliferation and concentration of IL-12 were performed using a one-way anova on the raw data, and the analyses for ELISPOT, total lesion scores and bacterial load results were performed using the rank sum test.