Multiskan go microplate

Nitrophenol phosphate (NPP) quantitative ALP assays were performed to assess osteogenesis in the C2C12s seven days after transfection. Cells were washed with PBS then lysed with 100ul lysis solution (0.5% Triton X-100 in deionized water) per well with 250RPM radial shaking for 1 h. 10ul from each well was transferred to a new 96 well plate, then a 90ul of NPP working solution (5mM NPP (4876, EMD Millipore), 0.5 M 2-AMP (A9199, Sigma Aldrich), 2mM MgCl2 (25108.260, VWR chemical), pH 10.3 in deionised water) was added to each well. A 4-Nitrophenol (4NP) end-product standard curve of 10–200µM (4NP to concentration(1048, Sigma Aldrich), 0.5 M 2-AMP, 2mM MgCl₂, pH 10.3 buffer) was run simultaneously to allow calculation of the final 4NP concentration in each well. Plates were incubated at room temperature for 45 min then absorbances at 405 nm and a 600 nm wavelength control were measured using a microplate spectrophotometer (Multiskan GO microplate reader, Thermo Fisher). ALP assays were performed in technical triplicate (three wells per sample). Mean values were used for analysis.

Chip-based hollow microcapsule production was performed as described previously^{54 (link),73 (link)}. In short, a polymethyl methacrylate (PMMA) microfluidic chip was used in a flow-focusing setup to produce droplets consisting of 5% Dex-TA and 250 U/ml HRP within an oil phase of n-hexadecane with the addition of 1% (v/v) Oil-red-O and 1% span 80 as a surfactant. The resulting emulsion was broken by three consecutive washes using n-hexadecane without surfactant and a subsequent wash with phosphate-buffered saline (PBS). Absorption of n-hexadecane with and without Oil-red-O and hollow microcapsules produced using chip-based microfluidics or IAMF was measured using spectrophotometry (Multiskan Go Microplate; Thermo Scientific) at 512 nm.

Antioxidant activities were determined by previously reported methods [20 (link)] using 96-well plates. Absorbance was recorded on Multiskan^® Go microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA) and standard curves with R² ≥ 0.99 were constructed with standard solutions. Results were reported on dry weight basis.

Plasma total antioxidant status (TAS) levels were quantified using 2,2′-azino-bis (3 ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Randox Laboratories Ltd., Antrim, UK), which is incubated with a peroxidase to produce the radical cation ABTS+. The bluish green staining of the ABTS+ cation is relatively stable and measured at 600 nm; antioxidants present in the plasma cause suppression of this color production to a degree that is proportional to the concentration. The kinetics reaction was measured with a spectrophotometer Multiskan Go Microplate (Thermo Scientific, Denver, CO, USA).

Bifidobacterium infantis BCRC14602, Bifidobacterium adolescentis BCRC14606, Bifidobacterium bifidum BCRC14615, Bifidobacterium longum BCRC14634, Bifidobacterium breve BCRC11846, Lactobacillus rhamnosus GG BCRC16000, Lactobacillus delbrueckii subsp. bulgaricus BCRC10696, Lactobacillus plantarum BCRC11697, Lactobacillus acidophilus BCRC14079, Streptococcus salivarius subsp. thermophiles BCRC14085 were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu city, Taiwan). All LAB strains were grown in MRS medium (Sigma-Aldrich, MI, USA). For storage, stock cultures were kept in 20% glycerol at -80°C. Viable cells were grown in MRS medium at 37°C for 20 hours as inoculum and sub-cultured twice a month [16 (link)]. The standard growth curve was measured at 600 nm using a Multiskan GO microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Growth inhibition assays were performed as previously described with a slight modification. Briefly, a culture (5 mL) of bacteria strains were grown from a single colony for 8 h at 37 °C in Lysogeny Broth (LB) media and diluted back to an OD₆₀₀ of 0.001. A 96-well plate assay was set up with 90 μL of the respective cultures in LB-media either with DMSO or containing 10–100 μg mL⁻¹ of the compounds. The experiment was repeated with and without 2 μg mL⁻¹ of the 10-nm nanoparticles. The OD₆₀₀ for all wells was recorded on a Multiskan™ GO microplate spectrophotometer (Thermo Scientific™, Waltham, MA, USA) at 0, 8, 16 and 32 h. To assure there were no absorbance contributions from the inhibitors, a change in OD₆₀₀ was determined by the subtraction of the absorbance from the blank containing an equivalent concentration of inhibitor. The concentration of the compound that inhibited 50% of the microorganism growth was calculated using the GraphPad Prism version 7.02.

Cell viability was assessed by the methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay according to manufacturer's instructions. The absorbance was measured at 570 nm using a Multiskan™ GO microplate reader (Thermo Fisher Scientific).