Ckx31 microscope

Two-fold dilutions of studied compounds TO1-biotin and 4a–d and DMSO as negative control were prepared in cultural medium (FSBSI ‘Chumakov FSC R&D IBP RAS’, Russia). Cell suspensions were added to the wells with compounds dilutions or DMSO control (∼2 × 10⁴ cells per well). The final concentration series of eight dilutions started from 50 μM. The cells were incubated at 37°C in a CO₂-incubator for 24 h and 7 days. After incubation, the cells were analyzed using Olympus CKX-31 microscope for cell viability. For this, cultural medium was substituted with resazurin solution (25 mg/mL) and cells were incubated at 37°C in a CO₂-incubator for 4 h. Then 20 ml of 10% SDS was added to stop the reaction and fluorescence was measured with Promega GloMax-Multi Detection System at λ_ex 525 nm and λ_em 580–640 nm. As additional controls, the same series of cells treated with compounds or DMSO dilutions but without resazurin solution to subtract the background fluorescence were used; and a medium with resazurin solution was used to set up a minimal value of non-reduced resazurin. All experimental procedures were performed in two replicates. Statistical analysis was performed, and fluorescence curves were plotted using MS Excel 2013. The 50% cytotoxic concentration (CC₅₀) was calculated (compound concentration required to induce a cytopathic effect in 50% of the cells in a monolayer).

The median tissue culture infective dose (TCID50) assay was used to determine the multiplicity of infection (MOI) of the virus in RD cells. For cell infection, when RD cells grew to 80% in a 10 cm dish, EV-D68 was added at an MOI of 0.1. The cytopathic effect (CPE) caused by EV-D68 infection was observed by light microscopy at different hours post infection (hpi) using an Olympus CKX31 microscope (Olympus). Working stocks (1*10^6.25 PFU per ml) were stored at 80°C.

Following fixation in 4% paraformaldehyde for 24 h, the intestinal segments were embedded in paraffin and 5 μm sections were sectioned with a rotary microtome. Then, the sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Olympus, Tokyo, Japan). Photomicrographs were taken with an Olympus CKX31 microscope (Olympus, Tokyo, Japan). The morphometric analysis was performed on 10 randomly-selected, well-oriented villi and crypts per piglet. A computerized microscope-based image analyzer (Olympus dotslide Virtual Slide System, Tokyo, Japan) was used to determine the height of villus (from the tip of the villus to the villus-crypt junction) and crypt depth (from the crypt-villus junction to the base of the crypt).

Trophozoites were cultured on a glass slide in a humid chamber at 37°C for 1 h, and the morphology was observed by phase-contrast microscopy (LCAch N 20×/0.40 PHP ∞/1/FN22 objective and CKX31 microscope; Olympus, Tokyo, Japan). The percentage of flagellate or amoeboid forms was measured from 600 trophozoites within 12 random microscopic fields. Compared to flagellate trophozoites, which have a solid spherical shape and a diameter of <10 μm, trophozoites with a stretching diameter of >10 μm and a morphology transforming into a distinctive irregular appearance or a flat round-disk form lying on the glass surface are defined as the amoeboid form of T. vaginalis trophozoites.