May gr nwald giemsa reagents

Lungs were filled thoroughly with 1 ml aliquots of pyrogen-free saline through a 22-gauge bead-tipped feeding needle introduced into the trachea. The lavage fluid was collected in a plastic tube on ice and centrifuged at 400 g, 4°C, for 5 min. For differential BAL cell counts, cytospin preparations were made and stained with May- Grünwald Giemsa reagents (Sigma-Aldrich). At least 200 cells per cytospin preparation were counted and the absolute number of each cell type was calculated. Photographs were observed using a BX51 microscope (Olympus, Milan, Italy) and images were captured using a high-resolution DP71 camera (Olympus).

Murine experiments were performed according to the Italian Approved Animal Welfare Authorizations 360/2015-PR (date of approval 15 May 2015) lasting for five years (2015–2020) and Legislative decree 26/2014 regarding the animal license, obtained by the Italian Ministry of Health. Four- to six-week-old C57BL/6 mice were purchased from Charles River (Calco, Italy). Breeding pairs of CF mice homozygous for the F508del-CFTR that had been backcrossed for 12 generations to the C57BL/6 strain, or in the FVB/129 outbred background (Cftrtm^1EUR, F508del, abbreviated Cftr^F508del mice), were obtained from Bob Scholte, Erasmus Medical Center Rotterdam, The Netherlands [18 (link)] and maintained in our animal facility. Mice were treated as described below and monitored for fungal growth, expressed as log10 CFU per organ, mean ± SD. Bronchoalveolar lavage (BAL) was performed by cannulating the trachea and washing the airways with 3 × 0.5 mL of PBS to collect the BAL fluid. Total and differential cell counts were done by staining BAL smears from allergic mice with May-Grünwald Giemsa reagents (Sigma-Aldrich, St. Louis, MO, USA) before analysis.