Sepmate 50 system

Human macrophages were isolated from whole blood using the SepMate-50 system (Stemcell Technologies, Seattle, WA) and following a University of Arizona IRB-approved protocol with written consent from the donors. After isolation, the cells were cryopreserved gradually with a controlled rate freezer. For experimental use, cryopreserved peripheral blood mononuclear cells (PBMCs) from normal (healthy) adult controls were placed in complete RPMI (RPMI, 10% fetal bovine serum, penicillin/streptomycin, and DNase [3 ng/ml]) and thawed to 37°C. Cells were collected by centrifugation at 500 × g for 6 min, resuspended in X-VIVO-15 (Lonza, Walkersville, MD), a serum-free medium, plated into 96-well plates (Fisher Scientific) (10⁵ cells in 100 µl medium/well), and placed in an incubator overnight at 37°C with 5% CO₂. The following day PBMCs were washed with serum-free medium and resuspended in fresh medium, human MCSF (Sigma-Aldrich, St. Louis, MO) was added to each well (1 ng/ml), and cells were incubated for 5 days at 37°C with 5% CO₂. Experiments were also carried out in four-well slide chambers (Bio Express, Visalia, CA) with the same medium and additives and 200 µl/well. Slide chambers allowed for better photomicroscopy of the macrophage-fungus interactions.

Human monocytes were isolated from whole blood using the SepMate-50 system (Stemcell Technologies, Seattle, WA, USA). The protocol was approved by the University of Arizona IRB. Cells were then cryopreserved. Peripheral blood mononuclear cells (PBMCs) from normal (healthy) adult controls were placed in complete RPMI (RPMI, 10% fetal bovine serum, penicillin/streptomycin, and DNase (3 ng/mL)) and thawed to 37 °C. Cells were collected by centrifugation, resuspended in X-VIVO-15 (Lonza, Walkersville, MD, USA), a serum-free medium, plated into 96-well plates (Fisher Scientific) (10⁵ cells in 100 μL medium/well), and placed in an incubator overnight at 37 °C with 5% CO2. On the following day PBMCs were washed with serum-free medium and resuspended in fresh medium, human Macrophage Colony Stimulating Factor (Sigma-Aldrich, St. Louis, MO, USA) was added to each well (1 ng/mL), and cells incubated for 5 days at 37 °C with 5% CO₂ [5 (link)]. Rat bone marrow macrophages (ScienCell Research Laboratories, Carlsbad, CA, USA) were treated in the same manner with the exception that 10% rat serum was provided in the RPMI as per manufacturer’s directions.