Multiskan spectrum

Overnight cultures of bacteria (1 × 10⁶ CFU/mL) grown in BHI broth were diluted 1:100 into fresh BHI broth. Bacteria were cultured in 96-well flat bottom polystyrene microtiter plates at 37°C. Cell growth was monitored with a Multiskan Spectrum (Thermo, Multiskan Go, United States), and the OD₆₀₀ was measured in 1 h intervals. Each analysis was performed in triplicate. The representative growth curves are plotted in the figure.
For CFU counts, after diluted, the bacteria were cultured at 37°C for 1, 4, 8, and 12 h. Then bacterial suspension was serially diluted in BHI and plated on BHI agar plates. CFU values were calculated after the plates were incubated anaerobically at 37°C for 48 h. In order to see how growth looked like under different carbohydrates, the overnight bacteria (1 × 10⁶ CFU/mL) grown in BHI broth were diluted 1:100 into the tryptone-vitamin (TV) base medium (3.5% tryptone with 0.04 μg of p-aminobenzoic acid/ml, 0.2 μg of thiamine-HCl/ml, 1 μg of nicotinamide/ml, and 0.2 μg of riboflavin/ml) which was supplemented with either 10 mM (limiting) or 100 mM (excess) sucrose, glucose, or lactose (Moye et al., 2014c (link)). Cell growth was monitored with a Multiskan Spectrum (Thermo, Multiskan Go, United States), and the OD₆₀₀ was measured in 1 h intervals. Each analysis was performed in triplicate. The representative growth curves are plotted in the figure.

Interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) concentrations were measured in sera from participants at baseline using ELISA kits (Diaclone, Besancon, France), according to the manufacturer’s instructions. Absorbance at 450 and 630 nm were measured using a scanning multiwell spectrophotometer (Multiskan spectrum, Thermo-Fisher Scientific, Waltham, MA, USA). Absorbance at 630 nm was subtracted from that at 450 nm to correct optics imperfections. Finally, CRP was measured using ELISA kits (Proteintech, Manchester, UK) according to the manufacturer’s instructions. Samples with different concentrations of standards were analyzed to obtain calibration curves. Adiponectin, leptin and ceruloplasmin concentrations were measured in sera at 0, 2 and 4 h postprandially, using ELISA kits (AssayPro, St. Charles, MO, USA) according to the manufacturer’s instructions. Absorbance at 450 and 570 nm were measured using a scanning multiwell spectrophotometer (Multiskan spectrum, Thermo-Fisher Scientific, Waltham, MA, USA). Absorbance at 570 nm was subtracted from that at 450 nm to correct optics imperfections. Samples with different concentrations of standards were analyzed to obtain calibration curves.

Antioxidant activity of SOD was measured based on the procedures of Cayman’s superoxide dismutase assay kit which is purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Basically, xanthine oxidase can react with the hypoxanthine so as to generate the superoxide radical (O₂^•−). The superoxide radical has interacted with the tetrazolium salt and the enzyme activity of SOD was measured via the instrument of spectrophotometry (Thermo Scientific Multiskan Spectrum, Vantaa, Finland). The SOD activity was expressed in terms of unit per mg of protein concentration. On the other hand, the CAT levels were assayed by catalase commercial kit purchased from Cayman Chemical Company (Ann Arbor, MI, USA). In brief, this analytical procedure is that the methanol reacts with hydrogen peroxide under the catalyzation of the CAT enzyme so as to generate the formaldehyde. Finally, the chromogen of 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole was reacted with the formaldehyde and the CAT levels were measured via spectrophotometer (Thermo Scientific Multiskan Spectrum, Vantaa, Finland), and enzyme activities were expressed in terms of U per mg of protein concentration.

Total phenolic compounds were determined following the method of Maphosa and Jideani (4 (link)). Samples (250 mg) were mixed with 10 mL of distilled water and 1 mL of H₂SO₄ in 14-mL centrifuge tubes and then incubated at 80 °C for 20 h before centrifugation (4000×g, 5 min, 21 °C). The supernatant was diluted tenfold and analysed in a 96-well plate using the Folin-Ciocalteu assay by mixing 25 µL sample with 125 µL of 0.2 M Folin-Ciocalteu reagent and 100 µL of 7.5% Na₂CO₃ solution. The mixtures were left to stand for 2 h in the dark. Absorbance was then measured with a spectrophotometer (Multiskan Spectrum; Thermo Fisher Scientific) at a wavelength of 765 nm using a gallic acid standard calibration curve. The results were expressed as mg of gallic acid equivalents (GAE) per g of dry extract.
Ferric reducing antioxidant power (FRAP) assay was conducted following the method of Maphosa and Jideani (4 (link)) with vitamin C as a standard. Samples (250 mg) were diluted tenfold and then mixed with 0.3 mL FRAP reagent (30 mL acetate buffer, 3 mL FeCl₃, 3 mL 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) and 6 mL H₂O). The mixture was poured into a 96-well plate, left to stand for 30 min and then read in a spectrophotometer (Multiskan Spectrum; Thermo Fisher Scientific) at a wavelength of 593 nm. The results were expressed as µmol of ascorbic acid equivalents per g of dry extract.

The cytotoxicity of free DOX, blank PMs, and various DOX-PMs against HepG2 cells were evaluated by standard MTT assay [36 (link),37 (link),38 (link),39 (link)]. In brief, HepG2 cells were seeded into a 96-well plate at an initial density of 1 × 104 cells/well in 200 μL DMEM medium and cultured in incubator for 24 h. The medium was removed, and 200 µL/well of free DOX, blank PMs, and DOX-PMs with different concentrations of DOX were added and cultured for 24 h. The wells without cells were used as blank, and the wells with cells but without treatment were used as control. After addition of 20 µL of MTT solution, the plate was shaken for 5 min at 150 rpm and then cultured for 4 h in incubator. After discarding the culture supernatants, 200 µL of DMSO were added to each well. The plate was gently agitated for 15 min, and the absorbance of sample was recorded by a microplate reader (Multiskan Spectrum, Thermo Scientific, Vantaa, Finland) at 490 nm. The cell viability (%) was defined as the absorbance ratio of difference between sample and blank and difference between control and blank.

Absorbances were measured using a scanning multi-well spectrophotometer (Multiskan spectrum, Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence intensity was measured by a FACSCanto II flow cytometer (Bd Biosciences, San Jose, CA, USA) and calibrating using FACSCanto II analyzer software (Bd Biosciences, San Jose, CA, USA). Gene expression was determined by means of a CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA). DMEM medium, fetal bovine serum (FBS), penicillin, streptomycin, and trypsin were purchased from Biowest (Nuaillé, France). Lipopolysaccharide (LPS) (from E. coli 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The total protein in each batch of AMEED was assessed
using a standard Bradford protein assay. Briefly, 20 µl of
each sample and a diluted standard that contained 10 µg/
µl .-globulin were added to the wells of a 96-well plate
(in duplicate), followed by the addition of 500 µl Bradford
buffer (5000006, Bio-Rad Laboratories, Inc., Hercules,
CA, USA) to each well and mixed. The optical density
at 595 nm was then measured using a spectrophotometer
(Multiskan Spectrum, Thermo Fisher Scientific Oy,
Vantaa, Finland).
The concentrations of EGF, KGF, hepatocyte growth
factor (HGF), and interleukin-1 receptor antagonist (IL1RA),
as important amniotic membrane proteins necessary
for epithelial regeneration (14 (link)), were assessed using
commercially available enzyme-linked immunosorbent
assay (ELISA) kits (Catalogue no.: DEG00, DKG00,
DHG00, and DRA00B, R & D Systems Inc., Minneapolis,
MN, USA) according to the manufacturer’s protocols.
Four batches of AMEED were used for this growth factor
analysis. The stability of the growth factors was tested
after one month to one year of storage at -70°C, after 7
days of storage in a refrigerator (2-8°C), and after 2 days
of storage at room temperature.