Counting chamber

Fresh samples of human fetal adrenal glands or gonads were placed in RPMI 1640 medium (C11875500BT, Gibco), which contained 5% fetal bovine serum (FBS; SH30070.03, HyClone) and 1% penicillin–streptomycin (15140-122, Gibco), and quickly transported to the laboratory on ice. Then, the fetal adrenal glands and gonads were separated under a stereomicroscope (Nikon), washed with cold D-PBS (311-425-CL, Wisent) and sliced into approximately 1–2-mm³ pieces. The tissues were transferred to digestion solution [0.1 mg/ml Liberase TL (5401020001, Roche) and 1 mg/ml DNase I (10104159001, Roche) in RPMI 1640] at 37°C with gentle shaking throughout (adrenal glands for 20 min and gonads for 30 min), filtered through a 100-μm cell strainer (352360, Falcon), centrifuged and resuspended in 5 ml of 1X red blood cell (RBC) lysis buffer (420301, BioLegend) for 5 min on ice. Then, the cells were washed twice with D-PBS containing 1% FBS, filtered through a 40-μm cell strainer (352340, Falcon), centrifuged and resuspended in DPBS with 1% FBS. The cell number and viability were assessed by Trypan blue (152 50-061, Gibco) staining and counting in a counting chamber (717805, Brand).

The I. fumosorosea isolate originated from the horse chestnut leaf miner, Cameraria ohridella Decka and Dimic (Lepidoptera: Gracillariidae). The strain is deposited under number CCM 8367 as a patent culture in the Czech Collection of Microorganisms in Brno [39 ]. As a reference strain, we used an isolate cultured from the commercial product PreFeRal® WG (Biobest, Belgium; I. fumosorosea strain Apopka 97 as an active ingredient). Blastospores of both strains were obtained after 120 hours of submerged cultivation in growth media (glucose, maltose, starch and peptone) using an orbital shaker at 140 cycles per minute at 23°C. The number of blastospores in suspension was counted with a Bürker counting chamber (Brand, Wertheim, Germany) and adjusted to required concentration. The soaking agent Tween 80 (Sigma-Aldrich) was added to the suspension at a concentration of 0.02% (v/v).