Spark 10m

The DPPH radical scavenging activity of the cassava gari was carried out according to the method described by Feregrino-Pérez et al. [18 (link)] with some modifications. To the sample solution (40 μL), 150 μL of DPPH (1 mM) was added in a 96-well microplate (New York, USA). The solution was mixed for 40 s using multimode reader (Tecan SPARK 10M, V1.2.20, Austria) and incubated for 40 min in the dark place. After incubation, the absorbance was recorded at 517 nm with multimode reader (Tecan SPARK 10M, V1.2.20). The results were evaluated from the Trolox as a standard.

The DPPH radical scavenging activity of the cassava gari was carried out according to the method described by Feregrino-Pérez et al. [18 (link)] with some modifications. To the sample solution (40 μL), 150 μL of DPPH (1 mM) was added in a 96-well microplate (New York, USA). The solution was mixed for 40 s using multimode reader (Tecan SPARK 10M, V1.2.20, Austria) and incubated for 40 min in the dark place. After incubation, the absorbance was recorded at 517 nm with multimode reader (Tecan SPARK 10M, V1.2.20). The results were evaluated from the Trolox as a standard.

Free and bound flavonoid contents were determined as described by Xiong et al. [16 (link)] with some modifications using multimode reader (Tecan SPARK 10M, V1.2.20, Austria). In brief, 20 μL of sample was mixed with 20 μL of 5% sodium nitrite and of 2% aluminum chloride. Then, the mixture was incubated for 3 min before adding 50 μL NaOH (0.5 M). The solution mixture was mixed again for 40 s and incubated for 6 min. Then, 30 μL of 2% sodium acetate was added in each well and incubated for 40 min in the dark place. The absorbance was recorded at 510 nm using multimode reader (Tecan SPARK 10M, V1.2.20, Austria). The quercetin was used as a standard.

Explants were exposed in triplicate for 2 or 4 h to complete medium (negative control) or seminal plasma (25%, 50% in complete medium), washed, weighed, and incubated in RPMI containing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) substrate (0.5 mg/mL, Sigma-Aldrich) for 3 h at 37 °C. After removal of the MTT solution, tissues were incubated in methanol overnight at room temperature in the dark to dissolve non-specific precipitates. Absorbance of the MTT-formazan product was measured at 570 nm (corrected at 630 nm) using a Spark plate reader (TECAN SPARK 10 M). Tissue viability was determined by dividing the absorbance by the dry weight of the explant. The effect of seminal plasma on tissue viability was calculated as the ratio between seminal plasma-treated explants and negative controls.
A suspension of fluorescein isothiocyanate-dextran (250 µg/ml FD4, 4 kDa; Sigma-Aldrich) was added to the apical surface of the sealed polarized explant and a tight Caco-2 monolayer treated, or not, with seminal plasma (25%, 50% in complete medium). After 2 or 4 h of incubation at 37 °C, the basolateral medium was sampled and FD4 quantified at 520 nm using a Spark plate reader (TECAN SPARK 10 M). The optical density was expressed as the percentage of the positive control (FD4 added to the basal medium at the beginning of the experiment).

Reactions (50 μl final volume) were carried out in protease reaction buffer (50 mM HEPES, pH 7.4, 75 mM NaCl, 0.1% CHAPS, 2 mM DTT) containing 50 μM Ac-DEVD-AMC, or Suc(oMe)-AAPV-AMC. Samples were measured using an automated fluorimeter (SPARK 10 M; TECAN) at wavelengths of 430 nm (excitation) and 535 nm (emission). For suc-FLF-sBzl assay, substrate was diluted to a final concentration of 300 μM in protease reaction buffer (50 mM HEPES, pH 7.4, 75 mM NaCl, 0.1% CHAPS, DTNB 300 μM). Cathepsin G hydrolyzes the synthetic substrate suc-FLF-sBzl with the release of the thiobenzyl group. The free thiobenzyl group reacts with DTNB [5,5′-dithiobis(2 nitrobenzoic acid) and produces a chromophore (TNB), which absorbs at 430 nm. Samples were measured by automated fluorimeter (SPARK 10 M; TECAN).

Native Fluc spectra were collected in solution using a Cary Eclipse Fluorescence Spectrophotometer (Agilent) with 10 nM Quantilum (Promega, Madison, WI; E1701) in 30 mM MOPS, 10 mM β-mercaptoethanol, 1 mM ATP pH 7.4, 1 mM potassium luciferin, 10 mM MgSO₄, and 1 mg/mL bovine serum albumin, as described previously^{101 (link)}. Cellular pH calibration was performed with stably transfected SV40:Luc U2OS cells at 37 °C in white 96 well plates (Corning™ 3917) on a Tecan Spark 10 M, alternately using 550–575 nm and 610–635 nm filters to calculate 560/620 nm ratio (Supplementary Fig. 2c, d) with 5 s integration times. At the depicted timepoint, 10 μL of 10 μM FCCP was automatically injected to give a final concentration of 1 μM FCCP. Longitudinal measurements were also performed on a Tecan Spark 10 M (Supplementary Fig. 2c, d), under the same conditions, measuring total bioluminescence from each well (1 s integration) in addition to emission at 550–575 and 610–635 nm from the same well (5 s integration). For these experiments cell media was buffered with 20 mM MOPS instead of bicarbonate, as described previously⁸⁸ . The pH of the various media was measured and adjusted at 37 °C with 1 M HCl or NaOH, and then adjusted to 342 mOsm with 1 M NaCl.