Hemocytometer

Cells were seeded at 12.5 × 10³ per well in 6-well plates (Thermo Fisher) in 2 mL of complete cell media or in the indicated solutions. Cells were placed in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C and allowed to grow, with counts starting on day two and ending on day six. Cell media was renewed on day three. Cells were dissociated using 0.25% trypsin plus 1 mM EDTA in balanced salt solution. The number of viable cells was then determined by counting with a hemocytometer (Sigma Aldrich) at room temperature after 1 : 1 dilution of the sample with 0.4% trypan blue solution (Sigma Aldrich). Fold increase of cells was calculated by dividing the number of cells with intact cell membranes by the number of cells initially plated.

SNU-387 cells were cultured in 12-well plate at about 5 × 10⁴ per well for cell growth assay and in 96-well plates at about 5 × 10³ per well for cell proliferation assay. Cells were transfected with siRNA targeting FAT4 or control siRNA for 24, 48, 72 h. Cells were observed under the phase contrast microscopy for changes in morphology and cell numbers at the designated time. For cell growth analysis, cells were trypsinized and diluted 1:1 with 0.4% trypan blue (sigma) and viable cells were counted with a hemocytometer (Sigma). For cell proliferation assay, 10 μl of Cell Counting Kit-8 solution was added into each well containing 100 μl culture medium and incubated for 2 h at 37 °C. The optical density value of each well was measured by absorbance at 450 nm in a microplate reader. Experiments were performed in duplicates.

L. pneumophila serogroup 1 strain Philadelphia, ATCC 33152, and A. polyphaga, ATCC 30461, were obtained from the American Type Culture Collection (ATCC), USA. Stationary-phase L. pneumophila cell suspensions were established according to Ariyadasa et al. (17 (link)).
A. polyphaga axenic cultivation was conducted following the ATCC protocol (“Acanthamoeba polyphaga [Puschkarew] Page | ATCC,” 2021). Trophozoites were cultured in a T-25 tissue culture flask (Nunc EasYFlask, Thermo Fisher Scientific, USA) that contained 5 mL of sterile peptone yeast extract glucose (PYG, ATCC medium 712) medium (pH 6.5) supplemented with additives (0.05 M CaCl₂, 0.4 M MgSO₄·7H₂O, 0.25 M Na₂HPO₄·7H₂O, 0.25 M KH₂PO₄, Na citrate·2 H₂O, and 0.005 M Fe (NH₄)₂(SO₄)₂·6H₂O; Sigma-Aldrich, USA) at 25°C for ~5 days, until a dense monolayer was formed on the bottom surface of the flask. Once optimal growth was achieved, the trophozoite monolayer was washed twice with 5 mL of Page’s amoeba saline (PAS) medium (0.142 g/L Na₂HPO₄, 0.136 g/L KH₂PO₄, 0.004 g/L MgSO₄·7H₂O, 0.004 g/L CaCl₂·2H₂O, 0.120 g/L NaCl; Sigma-Aldrich, USA) and harvested by vigorous agitation. The harvested trophozoites were transferred into a Falcon tube and washed twice with DFTW (600 × g, 5 min). The final concentration was adjusted to 10⁵ trophozoites/mL using a hemocytometer (Sigma-Aldrich, USA).

Spermatozoa were collected from the vas deferens of the mice and suspended in a human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA). Regarding sperm counts, sperm cells were immobilized by dilution in water and counted in duplicate by using a hemocytometer (Sigma-Aldrich, Saint Louis, MO, USA). For determining the percent motility, the sperm medium was diluted to 10⁶/mL by using HTF and spotted onto a glass slide. A total of 200 sperm cells (both motile and immotile) were counted in duplicate under a microscope to derive an average percent motility. To analyze the sperm morphology, 100 sperm cells were evaluated on average. Individual sperm cells were categorized as having normal or abnormal morphology (including head, neck and tail defects and immaturity) according to the World Health Organization criteria [3 ]. The midpiece of each sperm was stained using Mito Tracker conjugated with Alexa Fluor 488 (10 mg/mL) (Invitrogen, Carlsbad, CA, USA); 4,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining.

To evaluate the effect of the A‐PRF‐XBSM mixture exudate on cell proliferation, a cell counting method with a hemocytometer was used. Here, 100%, 20%, and 4% A‐PRF‐XBSM were used as the experimental groups. Complete medium was used as the positive control.
hPDLSCs were seeded into a 96‐well plate (10³ cells/well; Nunc, Denmark) and cultured for 24 h in complete medium at 37°C with 5% CO₂. After 24 h, the complete medium was substituted for the A‐PRF‐XBSM mixture exudate at different experimental concentrations. These cells were further cultured for 1, 3, 5, and 7 days. At each foreshowed time point, cells were extracted using 0.25% trypsin/EDTA (Sigma‐Aldrich, MO, USA) and dyed with trypan blue (Sigma‐Aldrich, MO, USA) for cell counting using a hemocytometer (Marienfeld, Germany).