Phenol red free rpmi 1640

The number and percentage of dead cells based on plasma membrane integrity of the adherent cell population was quantified by analysis of microscopy images. Cells in 96-well flat bottom plates (30,000 cells/well) were stained with 2 µg/ml propidium iodide (PI, Sigma-Aldrich) and 2 µg/ml Hoechst 33342 (H3570, Sigma-Aldrich) in 40 µl/well phenol red-free RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS and 2 mM L-Alanyl-L-Glutamine and incubated for 5 minutes at room temperature (RT). Cells were imaged using a Leica AF6000 LC fluorescence microscope (Leica Microsystems, Wetzlar, Germany) combined with a 10x dry objective. Total and dead cell numbers were quantified by respectively counting the nuclei and the number of PI-positive cells using Fiji/ImageJ software (83 (link)).

Blood mononuclear cells (BMCs) were isolated by gradient centrifuge method from whole blood samples. Briefly, the same volume of blood was overlaid onto Histopaque 1077 (Sigma-Aldrich, St. Louis, MO), and centrifuged at room temperature for 30 min at 400 × g. The layer of cells over Histopaque was collected and washed two times with PBS 1×. The isolated BMCs were re-suspended at the concentration of 2.5 × 10⁶ cell/mL in phenol red free RPMI1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10 % fetal bovine serum (HyClone, Logan, UT), and 1× Penicillin-Streptomycin Solution (HyClone, Logan, UT). BMCs were cultured in 48-well plates and each sample was treated with HEWL (Sigma-Aldrich, St. Louis, MO), or freeze dried whole yeast extract of Candida albicans (Greer Laboratories, USA) at the final concentration 20 μg/mL or with Concanavalin–A (ConA) at the final concentration of 5 μg/mL (Sigma-Aldrich, St. Louis, MO), for a total of 3 different treatments. In addition, one untreated sample was included as a control for each time point. Cells were harvested at 4 and 24 hours post incubation and stored in TRIzol (Ambion, Carlsbad, CA) at −80 °C.

A. fumigatus conidia ATCC 46645 (strain NCPF 2109) cryopreserved in liquid nitrogen and maintained in medium that contained 0.9% saline, 0.01% Tween, and 30% glycerol was thawed and spread onto solid potato dextrose agar medium (Neogen, MI, United States). The culture was incubated for 5–7 days at 37°C. Plates that contained mycelium of A. fumigatus were scraped with sterile PBS containing 0.05% Tween-20 (Bio-Rad, CA, United States), and the conidia were collected via filtration through a sterile nylon mesh with a porosity of 40 μm (BD Biosciences, NJ, United States). The conidia were pelleted by centrifugation (3150 g, 25°C, 15 min), resuspended in RPMI 1640 (phenol red-free) (Sigma, MO, United States), and counted using a Neubauer chamber and an optical microscope (Leica Microsystems, Wetzlar, Germany) at 40× magnification. The cell concentration was adjusted for the subsequent stimulation experiments. For a specific set of experiments, A. fumigatus conidia were fixed in 4% paraformaldehyde (PFO) for 30 min at room temperature, extensively washed with PBS (2 ml, three times), centrifuged (3160 g, 15 min) and resuspended in RPMI.