Candida albicans isolate (CIMR #5), a virulent and well-characterized isolate [eight citations given in Clemons et al. (2006)], stored at −80 °C, was plated on blood agar plates
(BAP) and incubated for 20 h at 35 °C. After passage of C. albicans on BAP, yeast growth was harvested into saline, pelleted by centrifugation (400 g, 10 min), and counted in a hemacytometer. Candida albicans was suspended in CTCM to the required concentration for challenging monocytes, neutrophils, or macrophages. The cells remain almost completely as yeasts during the brief exposure periods. Monocytes or peritoneal macrophages in duplicate cultures were treated with increasing concentrations of 3M-003 or IFN-γ, https://www.selleckchem.com/products/pci-32765.html or CTCM vehicle, 0.2 mL per well. After incubation for 20 h at 37 °C in a 5% CO2 incubator, the supernatants were aspirated and monocytes or macrophages were challenged with C. albicans in 0.2 mL of CTCM or CTCM+10% fresh mouse serum. Effector to target ratios (E : T) 10 : 1, 50 : 1, and 100 : 1
were tested; 100 : 1 was generally optimal for plating under these conditions. Following a 2–4-h incubation period at 37 °C, each culture was harvested by aspiration into distilled water and culture wells were washed 10 times with distilled water. Microscopic examination of harvested culture wells confirmed that the well contents were removed. Harvested material from each culture was plated on BAP in duplicate. Inoculated BAP were incubated at 35 °C for 20 h, and CFU per culture were calculated. 0.1 mL per well of SCH772984 106 neutrophils mL−1 CTCM in duplicate cultures were treated with increasing FER concentrations of 3M-003 or IFN-γ for 20 h at 37 °C in a 5% CO2 incubator. Following the incubation period, neutrophils were challenged in situ with 0.1 mL of C. albicans in CTCM for 2 h at 37 °C. Cultures were harvested and harvested material was plated on BAP as described above for monocytes and macrophages. After incubation of plated material on BAP for 20 h at 35 °C, colony counts were performed and CFU per culture was calculated. Blood
from BALB/c mice was collected by cardiac puncture into EDTA-containing vacutainer tubes. Blood was pooled, diluted 1 : 1 in phosphate-buffered saline, 3 mL of the mixture was layered over 3 mL of Accu-Paque (Accurate Chemical and Scientific Corp., Westbury, NY), and centrifuged (400 g) for 30 min. PBMC at the interface were collected into Hanks’ balanced salt solution and cells were pelleted by centrifugation (200 g, 7 min). The viability of PBMC was determined by trypan blue exclusion and PBMC were counted in a hemacytometer. PBMC were suspended in CTCM. Dilutions of 3M-003 in CTCM (0.25 mL) were prepared in flat bottom 48-well tissue culture plates (Costar). Sets (eight wells per set) of 3M-003 dilutions were inoculated with PBMC (0.25 mL per well of 4 × 106 mL−1) and cultures were incubated for 24 h at 37 °C and 5% CO2.