Triad series multimode detector

Approximately 1 × 10⁶ cells/mL were suspended in MEM media supplemented with 0.1% (v/v) BSA and incubated with 1 μg/mL of fluorogenic calcein-AM (Invitrogen) for 30 min at room temperature in the dark. Cells were then centrifuged at 1,500 rpm for 5 min, washed three times with MEM supplemented with 0.1% (v/v) BSA, and resuspended at the density of 8 × 10⁵ cells/mL with MEM containing 0.1% (v/v) BSA. Afterwards, 4 × 10⁴ cells were suspended in 49 mL of MEM before placing onto the filter (12-µm pore size) of 96-well ChemoTx^® chemotaxis system (Neuro Probe), which was pre-coated with Geltrex (Life Technologies). Cells were allowed to migrate from the filter to the bottom compartment containing 29 µL of MEM supplemented with 10% (v/v) FBS at 37°C for 6 h. The non-migrated cells on the top of the filter were gently removed with a paper towel before the fluorescence intensity generated from the calcein-labeled cells in the bottom compartment was measured with excitation and emission wavelength at 485 and 520 nm, respectively, in a Triad series multimode detector (Dynex Technologies).

The ChemoTx^® 96-well plate (Neuroprobe, Gaithersburg, MD, USA) was used to assess motility of transfected OVCAR-3 and OV-90 cells. Addition of an even spread of dried 0.6 µL Geltrex (Gibco, Waltham, MA, USA) diluted 1:1 with media (RPMI1640 + 0.1% BSA) on the filter membrane was used to determine invasion. Briefly, cells were labelled with calcein AM (Life Technologies, Mulgrave, VIC, Australia) after 30 min of incubation in the dark on a nutator. Excess calcein AM were removed by washing the cells twice with media (RPMI1640 + 0.1% BSA). Portions of 4 × 10⁴ cells were then pipetted onto each pore of the filter above a microplate containing wells prefilled with chemoattractant (10% FCS) and media (RPMI1640 + 0.1% BSA). Reverse pipetting was employed at every step to prevent bubble entrapment. After a 6-hour, 37 °C incubation, cells that had migrated or invaded the filter were measured using the Triad series multimode detector (Dynex Technologies, Chantilly, VA, USA) at 485–520 nm. Assays were carried out in biological triplicate and had technical replicates to a total of n = 21–24 per cell line per construct transfected. Statistical analysis was carried out on GraphPad Prism 8 first by Shapiro−Wilk test of normality followed by either Mann−Whitney U test (non-normal distribution) or unpaired t-test (normal distribution).

OVCAR-5 cell motility was assessed using a ChemoTx^® 96-well plate (Neuroprobe, Gaithersburg, MD, USA), as previously described [29 (link)]. Briefly, cells were labelled with calcein AM (1 µg/mL, Life Technologies, Mulgrave, VIC, Australia) for 30 min in the dark on a nutator. Excess calcein AM was removed by washing with media (RPMI1640 + 0.1% BSA). A concentration of 40,000 cells/50 µL were loaded onto uncoated 12 µm filter inserts. The cells were allowed to migrate for 6 h to the bottom well with chemoattractant (10% FBS) and media (RPMI 1640 + 0.1% BSA). Migrated cells were measured using the Triad series multimode detector (Dynex Technologies, Chantilly, VA, USA) at 485–520 nm. Assays were carried out in biological quadruplicate in 2–3 independent experiments.

Nitrite accumulation in the culture medium was considered to be an indication of NO production as described previously (Green et al. 1982 (link)). RAW 264.7 cells were seeded in 96-well plates at 1 × 10⁴ cells per well overnight. Cells were stimulated for 48 h with LTA from probiotic Bacillus strains at three concentrations (0.1, 1 and 10 µg/mL). 0.8 mL of Griess reagent (0.5 % sulfanilic acid, 1.4 % acetic acid, and 0.008 % naphtylethylenediamine) was mixed with culture supernatants. The optical density was determined at 535 nm (Libra S12, Biochrom, UK) by comparison with a NaNO₂ standard curve. A negative control with 5 mM sodium acetate buffer (pH 4.7) and two positive controls with commercial LPS from Escherichia coli K12 (InvivoGen, France) and commercial LTA from S. aureus (InvivoGen) were used.
LTA cytotoxicity was assessed by adding 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide solution (MTT, Sigma-Aldrich, USA) at a final concentration of 0.5 mg/mL in each well for 3 h at 37 °C in a humidified atmosphere containing 5 % CO₂. Afterwards, supernatants were removed and formazan crystals were dissolved with 200 µL DMSO. Absorbance was measured with a microplate spectrophotometer at 595 nm (TRIAD series Multimode Detector, Dynex, USA) and compared to controls (data not shown).

WRL68 cells were seeded in a 25 cm² flask at a density of 1.0 x 10⁶ cells/flask and allowed to grow for 24 h. The cells were then treated with H₂O₂ for 30 min and post-incubated for 2 h in fresh medium (Fig 1). After this, the cells were recovered, centrifuged at 300 g for 10 min and washed with PBS. Intracellular ATP was extracted using the boiling water procedure described by Yang and coworkers [34 (link)]: 400 μl of boiling water was added to each cellular pellet, which was subsequently vortexed and incubated for 5 min; the resulting suspension was centrifuged at 12000 g for 5 min. ATP content in the supernatant was determined using the Adenosine 5’-triphosphate (ATP) Bioluminescent Assay Kit (Sigma, cat # FLAA-1KT) according to the manufacturer’s instructions. Luminescence was measured from 96-well plates using a microplate reader (TRIAD series multimode detector, Dynex Technologies, Chantilly, VA) and the quantity of ATP was calculated using a standard curve. Proteins were measured using the Bradford method (Biorad). ATP content was expressed as nmol per mg of total protein.