Neubauer chamber

Peripheral blood mononuclear cells (PBMC) were isolated by Biocoll (Biochrom, Berlin, Germany) density gradient centrifugation and cryopreserved at minus 80 °C until the time of analysis. The viability of obtained cells was planned at >95%, as determined by trypan blue staining (Sigma Aldrich, Saint Louis, MO, USA). The viable cells were quantified in a Neubauer chamber (Zeiss, Oberkochen, Germany). Samples were analyzed at the moment of imatinib discontinuation (month 0) and at 3 months after withdrawal.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. The viability of PBMCs was always >95%, as determined by trypan blue staining. The viable cells were quantified in a Neubauer chamber (Zeiss, Oberkochen, Germany) and leftover cells were cryopreserved in liquid nitrogen.

Blood samples were collected in vacuum tubes containing an anticoagulant (0.004% EDTA) from the antecubital vein of the runners at rest. Neutrophils were obtained from whole blood diluted in phosphate-buffered saline (PBS, 1:1; pH 7.4 containing 100 mmol/L CaCl₂, 50 mmol/L MgCl₂) and carefully layered on histopaque (d = 1.077) gradient (43 (link), 44 (link)). The tubes were then centrifuged at 400 x g and 4°C for 30 min. The supernatant, rich in mononuclear cells, was separated. Neutrophils were prepared from the inferior sediment, and 10 mL lyse solution, containing 150 mmol/L NH₄Cl, 10 mmol/L NaHCO₃, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4, were added to promote lysis of contaminating erythrocytes. The preparation was homogenized and maintained in ice for 10 min to allow erythrocyte lysis. Afterward, the tubes were centrifuged at 400 x g and 4°C for 10 min, and this procedure was repeated to reduce residual erythrocyte. Cells were washed once with PBS. Neutrophils were then counted in a Neubauer chamber in an optical microscope (Carl Zeiss, Jena, Germany), according to the protocol used in our previous study (30 (link), 45 (link)).

To ensure equal loads of each fungal species for the inoculation, fungi were inoculated on 2% potato dextrose agar (PDA) supplemented with the antibiotic chloramphenicol (50 mg L⁻¹) to prevent bacterial contamination and incubated in growth chambers at a constant temperature of 24 °C in the dark until sporulation (7–10 days—depending on species). A spore suspension of each fungus was prepared by flooding 5 mL of sterilised saline solution (0.9% NaCl) with 0.1% (v/v) aqueous Tween 80 and gently scraped using a sterile glass stick. The spores were counted under an optical microscope (Carl Zeiss) with a cell-counting haemocytometer (Neubauer chamber: Assistant Bright Line). Before application, the spore concentration was adjusted to 10⁶ spores mL⁻¹.
Buckwheat grains were sterilised by autoclaving (121 °C, 15 min) in Systec VX-150 autoclave, and samples of 10 g were inoculated with 10 mL of fungal spore suspension in a 150 mL flask and vigorously shaken on a rotatory shaker (180 rpm) for 30 min to distribute fungal spores uniformly. After the inoculation, the grains were transferred to sterile Petri dishes, spread evenly, and let air dry for 24 h under a laminar flow hood.

Mononuclear cells from peripheral blood (PBMC) and bone marrow (BMMC) were isolated by Ficoll (Biochrom AG, Berlin, Germany) density gradient. Next, cells were washed twice in phosphate-buffered saline (Biochrom AG, Berlin, Germany) and counted. The viability of obtained PBMC and BMMC was always >95%, as determined by trypan blue exclusion (Sigma-Aldrich, Schnelldorf, Germany). The viable cells were quantified in a Neubauer chamber (Zeiss, Jena, Germany) and stored for RNA preparation at −192 °C in liquid nitrogen.