Neubauer hemocytometer

The semen analysis and preparation were performed as previously described by Punjabi 2018 et al. Patients were instructed to maintain 2–7 days of sexual abstinence. All semen samples were collected at the laboratory, and any missing ejaculate fraction was reported. Samples were weighed for volume and analysis was initiated within 60 min after ejaculation, including sperm concentration, using an improved Neubauer hemocytometer (Marienfeld GmbH, Lauda-Königshofen, Germany) combined with a positive displacement pipette (Microman, Gilson Inc., Middleton, WI, USA); sperm motility, including progressive and total motile sperms; and sperm morphology, adapting the modified Papanicolaou stain (Sigma-Aldrich Inc., St. Louis, MO, USA).

Conidial suspension of B. bassiana was prepared by scraping the fungal culture from 14 days old PDA medium into an Eppendorf tube containing 1 mL of autoclaved Triton X-100 (0.1%) solution (Duksan Pure Chemicals, Ansan, Korea). Conidia of the suspension were counted using a Neubauer hemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany) under 40× magnification.

Hemolymph was collected by cutting larval proleg and mixed with ACB (1:10, v/v). Hemocytes were counted using a Neubauer hemocytometer (Superior Marienfeld, Lauda-Königshofen, Germany) under a phase contrast microscope (BX41, Olympus, Tokyo, Japan) at 100 × magnification. Heat-killed (90 °C, 30 min) Escherichia coli (5 × 10⁵ cells/larva) and a test chemical (5-HT or SB-269970) were co-injected into hemocoel through abdominal proleg of L5 larvae in a volume of 5 µL using a 10 µL micro-syringe (Hamilton) after surface-sterilization with 70% ethanol. After 4 h of incubation at 25 ± 2 °C, hemolymph of the insect was collected and assessed for THC.

MCF10A cells were collected using Trypsin (ThermoFisher Scientific) and resuspended in Assay Media (same as MCF10A growth media, but with no EGF and 2% horse serum). At the same time, frozen Matrigel (Corning, New York, NY, USA) was spread evenly to each well of an 8-well chamber slide (Lab-Tek, Scotts Valley, CA, USA) and placed in a cell culture incubator for solidification. After counting the cells using a Neubauer hemocytometer (Marienfeld, Lauda-Königshofen, Germany), 5 × 10³ cells were resuspended in Assay Media containing 2% Matrigel (Corning) and 5% EGF (Peprotech) and were seeded to a well of a Matrigel-precoated 8-well chamber slide. Cells were allowed to grow in the incubator for 20 days, with fresh Assay Media containing 2% Matrigel and 5% EGF being added every 4 days. Images were captured every 5 days using an Inverted Primovert microscope (Zeiss).

Third-instar larvae of L. decemlineata were collected in potato kitchen gardens (Solanum tuberosum) near Karasuk town, Western Siberia (53°43′05.4″ N 77°38′14.4″ E). The larvae were maintained in a laboratory in 350 mL ventilated plastic containers (10 larvae per container) at 25 °C, 20% relative humidity, under a photoperiod of 14:10 light:dark. Foliage of S. tuberosum was used as feed.
Strain P-72 of M. robertsii (GenBank accession no. KP172147.2) was used, taken from the collection of microorganisms at the Institute of Systematics and Ecology of Animals, the Siberian Branch of the Russian Academy of Sciences. To obtain conidia, twice-autoclaved millet was inoculated with a 4-day-old submerged culture of P-72 and incubated for 10 days at 25 °C in the dark. Then, the culture was dried for 12 days at room temperature, and conidia were collected using a soil sieve and stored at 4 °C for 20 days until infection. For infecting the CPB, the conidia were suspended in an aqueous solution of Tween 20 (0.05%). Concentrations of the conidia were estimated by means of a Neubauer hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany).

PSMA-positive LNCaP cells (300265; Cell Lines Service) were cultivated in Dulbecco modified Eagle medium (DMEM)/Nutrition Mixture F-12 with GlutaMAX (1:1, DMEM-F12, Biochrom) supplemented with fetal bovine serum (10%, FBS Zellkultur) and kept at 37°C in a humidified CO₂ atmosphere (5%). A mixture of trypsin and ethylenediaminetetraacetic acid (0.05%, 0.02%) in PBS (Biochrom) was used to harvest cells. Cells were counted with a Neubauer hemocytometer (Paul Marienfeld).

Semen samples were collected before any medical intervention. Three to 5 days of sexual abstinence were asked of the participants before ejaculates were collected by masturbation. After liquefaction at room temperature, standard semen analysis was done according to the 5th WHO laboratory manual within 60 min of ejaculation by a trained technician [22 ]. After initial macroscopic assessment of the samples (color, viscosity, volume, and pH), microscopic assessment was performed under a bright-light microscope equipped with a contrast phase (DM 2000, Leica, Heerbrugg, Switzerland). The sperm concentration was estimated with an improved Neubauer hemocytometer (Paul Marienfeld, Lauda-Königshofen, Germany). Motility characteristics were evaluated using the standard grading system: progressive motility, nonprogressive motility, and immotility. Both eosin staining and hypo-osmotic swelling (HOS) tests were used to assess sperm viability. Sperm morphology was assessed according to Kruger’s strict criteria following Papanicolaou staining of the previously washed sperm smears. To distinguish peroxidase-positive leukocytes from peroxidase-negative round cells (other round cells), the LeucoScreen Kit (FertiPro N.V., Beernem, Belgium) was used.