Hemocytometer

For purifying 100,000–150,000 nuclei, 1 g of fresh tissue was harvested and ground in liquid nitrogen using a mortar and pestle to break the cell wall. To avoid enzymatic activity, all the following steps were performed at 4 °C. The resulting powder was transferred into 20 mL NPB (Nuclear purification Buffer containing 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA pH 8, 0.5 mM EGTA, 0.2 mM Spermine, 0.5 mM Spermidine and protease inhibitor × 1 (Sigma P2714)) and incubated on a rotator at 4 °C for 10 min. The sample was then transferred through a 40-µm mesh and centrifuged for 10 min at 1000 g and 4 °C. The pellet was gently resuspended in 7 mL NPBt (NPB supplemented with 0.1% Triton X-100). Nuclei were then bound to 25 µl of streptavidin-coated Dynabeads (Invitrogen, M-280 Strepavidin) according to the manufacturer’s protocol and washed with NPB. After washing 4–5 times with 1 mL NPBt, the nuclei were resuspended in 1 mL NPB. To count nuclei, a 20-µl suspension was loaded to a Marienfeld hemocytometer (Neubauer-improved, chamber depth of 0.1 mm, 0630010), and visualized with a Nikon Eclipse TS100 light microscope.

Trypan blue cell count was carried out to identify the amount of viable cells present in each sample. After 24 h incubation period, harvested cell suspension (10 μL) was added with equal volume of 0.4% trypan blue stain. Hemocytometer was used for cell counting under inverted light microscope (NIKON, Japan). Viable cells are those excluded from the stain.

L. plantarum ATCC 202195 and L. plantarum ATCC 14917 were purchased from the ATCC (Manassas, Virginia, USA). Genomic DNA from Lactobacillus crispatus strain VPI 3199 ATCC 33820DQ, Lactobacillus gasseri strain DSM 20243 ATCC 33323DQ, Lactobacillus iners strain AB107 ATCC 55195DQ, Lactobacillus acidophilus ATCC 4357D-5, Lacticaseibacillus casei ATCC 334D-5, and Limosilactobacillus reuteri strain F275 ATCC 23272D-5 was purchased from Cedarlane Labs (Burlington, Ontario, Canada).
L. plantarum ATCC 202195 and L. plantarum ATCC 14917 cells were cultured in De Man, Rogosa, and Sharpe (MRS) broth under anaerobic conditions at 37°C for approximately 20 hours. Prior to DNA extraction, L. plantarum ATCC 202195 cells were enumerated using hemocytometry. In brief, cells were subjected to two overnight growth passages in MRS broth at 37°C. A total of 100 mL of overnight culture was diluted 10 times in phosphate-buffered saline (PBS) before transferring 10 µL of cell suspension into the hemocytometer (Bright-line) and viewing the cells under ×40 magnification (Nikon Optiphot). Cells were counted from 20 different grid squares by three independent laboratory personnel before being averaged. Enumerated cell suspensions were centrifuged at 10,000 × g for 5 minutes, and the resulting cell pellets were stored at −80°C until DNA was extracted.

Growth was measured through the use of two techniques: counting the number of cells and/or measuring the fresh weight of a specific volume of the culture. Cell number was measured by using a Thoma hemocytometer and a light microscope (Nikon eclipse S5i). Fresh weight was acquired through gravitometric measurements. Starch was measured by using an enzymatic assay as described by Staudinger [105 ]. Total lipid content was measured as described by [7 (link)]. Chlorophyll content was monitored as described by [106 ]. The photosynthetic rate was measured with an imaging/pulse-amplitude modulation fluorimeter (OS1-FL, Opti-Sciences).

Six mice of each group were used to harvest BALF according to published protocol (Bracke et al., 2006 (link)). After stained with Wright-Giemsa dye, the total number of leukocytes and neutrophils in BALF were counted using a hemocytometer under optical microscope (Nikon, Japan).

C. vulgaris was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, and maintained in BG-11 medium. The algae were cultured at 24 ± 1 °C in an incubator under irradiance 81 µmol·m⁻²·s⁻¹ with a 14:10 (light:dark) photoperiod. Exponential growth phase algal cells were cultured in 150 mL culture medium in the presence or absence of PS-NPs with series of dilutions (0, 0.5, 2.5, 5, 10, 20, and 50 mg/L) in 250 mL Erlenmeyer flasks for 72 h. The initial cell density of algae was 1 × 10⁶ cells/mL. Three replicates were performed for each concentration. To ensure optimum growth, cultures were shaken five times per day during incubation. Algal cell density was monitored at 685 nm by UV spectrophotometer (UNICO 2802 S, Franksville, Racine County, WI, USA) as well as counted with hemocytometer under optical microscope (Nikon, Tokyo, Japan) per 24 h. The regression equation for the relationship between cell density (y × 1.0 × 10⁶ cells/mL) and absorption at 685 nm (x) was calculated as y = 31.31x + 0.12 (p < 0.01, R² = 0.99).
The algal growth inhibition ratio (IR) was calculated using the following equation:
$$\mathrm{I}{\mathrm{R}}_{\mathrm{i}}(\%)=({\mathrm{C}}_{\mathrm{c}\mathrm{i}}-{\mathrm{C}}_{\mathrm{t}\mathrm{i}})/{\mathrm{C}}_{\mathrm{c}\mathrm{i}}\times 100\%$$ where IR_i is the growth inhibition rate at time I, C_ci is the cell density of the control at time I, and C_ti is the cell density of the treated group at time i.

For each set of experiments, four cryopreserved semen straws (ejaculates) from four different stallions (200 × 10⁶ spermatozoa/0.5 mL straw) were used. These samples were obtained from GeneTec by Ativet (Pilar, Buenos Aires, Argentina; website: www.genetec.com.ar) and Los Pingos del Taita (Rio Cuarto, Córdoba, Argentina; website: www.lospingosdeltaita.com). Straws were thawed in a water bath at 37 °C for 30 s. Spermatozoa were selected using glass wool columns and washed by centrifugation at 600× g for 5 min at room temperature (RT). Pellets were resuspended in 100 μL of NCWM and sperm concentration and motility were examined using a hemocytometer mounted on a bright-field microscope stage heated at 38.5 °C at 100× magnification (Nikon Instruments Inc., Tokyo, Japan).