Lmc 3000

Human erythrocytes (O(I)+) were obtained from Vladivostok blood transfusion station. Erythrocytes were centrifuged with PBS (pH 7.4) for 5 min at 4 °C by 450× g on a LABOFUGE 400R (Heraeus, Hanau, Germany) centrifuge three times. Then, the residue of erythrocytes was resuspended in ice-cold PBS (pH 7.4) to a final optical density of 2.0 at 700 nm and kept on ice. For the hemolytic assay, 180 µL of erythrocyte suspension was mixed with 20 µL of Cucumarioside A₂-2 (positive control) and investigated compounds at concentrations range of 0.1, 0.5, 1, 5, 10 µM in V-bottom 96-well plates. After 1 h of incubation at 37 °C, plates were centrifuged at 900× g for 10 min on an LMC-3000 (Biosan, Riga, Latvia) laboratory centrifuge. Then, we carefully selected 100 µL of supernatant and transferred it to new flat plates, respectively. Lysis of erythrocytes was determined by measuring the concentration of hemoglobin in the supernatant with a microplate photometer Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm. The effective dose causing 50% of erythrocytes hemolysis (ED₅₀) was calculated using the computer program SigmaPlot 10.0. All experiments were made in triplicate, p < 0.01. ED₅₀ values are indicated in Table 4.

The pulverized R. rosea L. (1.00 g) was placed in a beaker flask, 20 mL of the different NADESs was added, shaken vigorously manually for a few seconds to form a slurry, and then the flask was placed in a temperature-controlled ultrasonic cleaner (Sapphire UZV-4.0, Moscow, Russia), at a sonication power of 50 W, a frequency of 35 kHz, and extracted for 60 min at 36 °C [62 (link)]. Then, 40% (v/v) aqueous ethanol was also used for comparison. After extraction was completed, the solutions were collected and centrifuged at 3000 rpm for 15 min in a table centrifuge (BIOSAN LMC-3000, Riga, Latvia). A supernatant of 5 mL volume was carefully removed, and a dilution of 5× was made. Finally, the diluted solutions were filtered through a 0.45 µm filter and analyzed through HPLC. These experiments were repeated three times.

Fecal samples were collected rectally from sheep naturally infected with Trichostrongylidae, during routine animal examination. Samples were transported to the laboratory in transport vials at the temperature of 4 ± 2 °C on the same day within 4–6 h for further examination. Parasite infection was confirmed by the modified McMaster method (eggs per gram of feces); 4 g of feces and 56 mL salt solution, and an egg suspension was prepared from the selected samples according to Coles et al. [19 (link)]. First, 20 g of feces was crushed in 200 mL of distilled water and was filtered through sieves (1000 µm, 100 µm, 50 µm, and 32 µm). The residue of the last sieve was collected with distilled water and was centrifuged (LMC-3000, Biosan, Latvia) for 10 min at 2000 rpm. The supernatant was discarded, and a salt solution (NaCl with a density of 1.018 g/mL) was added. The mixture was centrifuged for 15 min at 3000 rpm, and the supernatant was sifted through a 32 µm sieve. The eggs were collected from the sieve with distilled water.

Erythrocytes were isolated from human blood (A(+)) by centrifugation with phosphate-buffered saline (PBS) (pH 7.4) at 4 °C for 5 min by 450 g on a LABOFUGE 400R (Heraeus, Hanau, Germany) centrifuge three times. Then, the residue of erythrocytes was resuspended in ice cold phosphate saline buffer (pH 7.4) to a final optical density of 1.5 at 700 nm and kept on ice. For the hemolytic assay, 180 µL of erythrocyte suspension was mixed with 20 µL of test compound solution (including chitonoidoside A used as positive control) in V-bottom 96-well plates.
After 1 h of incubation at 37 °C, the plates were exposed to centrifugation 10 min at 900 g on a LMC-3000 (Biosan, Riga, Latvia) laboratory centrifuge. Then, 100 µL of supernatant was carefully selected and transferred to new flat-plates, respectively. Lysis of erythrocytes was determined by measuring the concentration of hemoglobin in the supernatant with a microplate photometer Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA), λ = 570 nm. The effective dose causing 50% hemolysis of erythrocytes (ED₅₀) was calculated using the computer program SigmaPlot 10.0. All the experiments were made in triplicate, p < 0.01.

Erythrocytes were isolated from human blood (AB(IV) Rh+) by centrifugation with phosphate-buffered saline (PBS) (pH 7.4) at 4 °C for 5 min by 450 g on centrifuge LABOFUGE 400R (Heraeus, Hanau, Germany) three times. Then, the residue of erythrocytes was resuspended in ice cold phosphate saline buffer (pH 7.4) to a final optical density of 1.5 at 700 nm, and kept on ice. For the hemolytic assay, 180 µL of erythrocyte suspension was mixed with 20 µL of test compound solution (including chitonoidoside L used as positive control) in V-bottom 96-well plates. After 1 h of incubation at 37 °C, plates were exposed to centrifugation for 10 min at 900 g on laboratory centrifuge LMC-3000 (Biosan, Riga, Latvia). Then, 100 µL of supernatant was carefully selected and transferred in new flat-plates, respectively. Lysis of erythrocytes was determined by measuring the concentration of hemoglobin in the supernatant with microplate photometer Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA), λ = 570 nm. The effective dose causing 50% hemolysis of erythrocytes (ED50) was calculated using the computer program SigmaPlot 10.0. All the experiments were carried out in triplicate, p < 0.01.

A total of 1 × 10⁵ isolated milk PMNs were seeded into a 96-well flat-bottom plate in duplicate and later incubated with PBS or serial dilutions of quercetin hydrate (13, 25, 50, and 100 μM) or curcumin (32, 65, 163, and 325 μM) in RPMI-1640 medium at 37 °C with 5% CO₂ for 45 min. After incubation, the plate was spun at 1200 rpm for 3 min (LMC-3000, BioSan, Riga, Latvia) and then the supernatant was discarded. All wells were infused with 2 μg/mL 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) in PBS [3 ]. After 15 min incubation, optical density (OD) of colored formazan was measured at OD₅₇₀ using an automated microplate reader (Anthos Labtec Instruments, Wals, Austria). Percentage of cell viability was quantified by the following equation: % viable cells = (OD _sample − OD _blank) × 100 (OD _control − OD _blank)

Erythrocytes were obtained from human blood (AB(IV) Rh+) by centrifugation with phosphate-buffered saline (PBS) (pH 7.4) at 4 °C for 5 min three times at 450 g on a centrifuge LABOFUGE 400R (Heraeus, Hanau, Germany). Then, the erythrocytes residue was resuspended in ice-cold phosphate saline buffer (pH 7.4) to a final optical density of 1.5 at 700 nm and kept on ice [22 (link)]. For the hemolytic assay, 180 µL of erythrocyte suspension was mixed with 20 µL of test compound solution (including chitonoidoside L used as a positive control) in V-bottom 96-well plates. After 1 h of incubation at 37 °C, the plates were exposed to centrifugation for 10 min at 900 g in a laboratory centrifuge LMC-3000 (Biosan, Riga, Latvia) [22 (link)]. Then, 100 µL of supernatant was carefully decanted and transferred into new flat plates. The erythrocyte lysis values were measured by measuring the supernatant’s hemoglobin concentration with a microplate photometer Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA), λ = 570 nm [23 ]. The effective dose causing 50% hemolysis of erythrocytes (ED50) was calculated with the computer program SigmaPlot 10.0. All the experiments were carried out in triple repetitions, p < 0.01.