Synergy ht microplate reader

Total serum protein was determined by the Bradford method using bovine serum albumin (BSA) as the external standard. The serum samples were diluted 200-fold before analysis. The absorbance was measured at 595 nm on a Biotek Synergy HT Microplate Reader (Agilent, Santa Clara, CA, USA). The determination of total polyphenols and creatinine in urine has been described in a previous publication of our group [7 (link)], performing a standard curve with commercial gallic acid from 300 to 4.7 µg/mL in ultrapure water. To normalize the data obtained for the polyphenols, we determined the concentrations of urine creatinine by a colorimetric reaction. Urine samples were diluted 20-fold and 10 µL pipetted into a 96-well, flat-bottom plate. Next, 200 µL 0.1% picric acid and 15 µL of NaOH were added. After a 15-min incubation, the plate was read at an absorbance of 500 nm by using a Biotek Synergy HT Microplate Reader.

The viability and proliferation of hADSCs cultured in the CHI and CHI/HA scaffolds were evaluated by means of a water-soluble tetrazolium-1(WST-1) reagent (Roche, Basel, Swiss) in combination with Quant-iTTM PicoGreen dsDNA Kit (Invitrogen, California, USA) at day 1 and subsequently at day 7, 14, or 28, as described previously [31 ]. Briefly, the CHI and CHI/HA scaffolds with the hADSCs were transferred to a new 24-well plate and washed twice with PBS after which 0.5 ml fresh normal growth medium containing WST-1 at 10:1 (v/v) was added to each well and incubated for 3 h at 37 °C at 5% CO₂. The absorbance of the WST-1/medium mixture was read at 450 nm using a Synergy HT microplate reader and Gen 5 2.03 software (BioTek, Vermont, USA) in a 96-well plate. The same scaffolds were used for the PicoGreen dsDNA Assay. Here The 1% PSCs were washed twice with PBS. According to the manufacturer’s protocol, the cells were lysed from the scaffold and DNA standards were mixed with TE-buffer and subsequently with Quant-iT PicoGreen dsDNA reagent. The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a Synergy HT microplate reader and Gen 5 2.03 software (BioTek).

The increase in cell numbers with time indicating proliferation and the metabolic activity of hADSCs cultured in the CHI and CHI/HA scaffolds were evaluated by means of Quant-iT^TM PicoGreen dsDNA Kit (Invitrogen, Carlsbad, CA, USA) and a water-soluble tetrazolium-1(WST-1) reagent (Roche, Basel, Swiss), respectively, at Day 1 and subsequently at Day 7, 14, or 28. Briefly, the CHI and CHI/HA scaffolds with the hADSCs were transferred to a new 24-well plate and washed twice with PBS, after which 0.5 mL of a fresh normal growth medium containing WST-1 at 10:1 (v/v) was added to each well and incubated for 3 h at 37 °C at 5% CO₂. The absorbance of the WST-1/medium mixture was read at 450 nm using a Synergy HT microplate reader and Gen 5 2.03 software (BioTek, Winooski, VT, USA) in a 96-well plate. The same scaffolds were used for the PicoGreen dsDNA Assay. Here, the 1% PSCs were washed twice with PBS. According to the manufacturer’s protocol, the cells were lysed from the scaffold and DNA standards were mixed with TE-buffer and subsequently with Quant-iT PicoGreen dsDNA reagent. The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a Synergy HT microplate reader and Gen 5 2.03 software (BioTek).

Serum free fatty acids were measured in duplicate using a NEFA-HR kit (Wako chemical GmbH, Neuss, Germany) according to the manufacturer’s instructions. In short, 5 µl serum was incubated with 200 µl reagent R1 for 10 min at 37 °C, which was subsequently incubated with 100 µl reagent R2 for 10 min at 37 °C, after which absorbance at 546 nm was measured using a synergy HT microplate reader (BioTek instruments Inc., Winooski, VT, USA) and corrected for background absorbance at 660 nm. Standard curves of NEFA standard solution (Wako chemical GmbH) were used for quantification of free fatty acids. Liver triacylglycerols were measured in triplicate using a triglyceride liquicolor mono kit (Human, Wiesbaden, Germany), according to the manufacturer’s instructions. In short, liver was ground in liquid nitrogen and homogenised in homogenization buffer (10 mM Tris, 2 mM EDTA, and 250 mM sucrose). 4 µl of homogenate was incubated with 100 µl of reagent for 45 min, after which absorbance at 500 nm was measured using a synergy HT microplate reader (BioTek instruments Inc., Winooski, VT, USA). Standard curves of triacylglycerol standard supplied in the kit was used for quantification of triacylglycerol levels. Triacylglycerol levels were expressed per amount of total protein in the homogenates, which was measured using the DC-protein kit (Bio-rad).

Quantification of NO accumulation in cell media was performed using the Griess Reagent (Sigma–Aldrich^®—Poole, Dorset, UK), as described before [10 (link)]. For analysis, 100 µL of cell culture medium of each condition were used and the manufacturer’s instructions followed. The absorbance (Abs) was read at 540 nm in a Synergy HT microplate reader (Biotek). For ELISA, cell media was collected from the plate and immediately frozen at −80 °C until analysis. TNF-α release was assayed by sandwich ELISA according to the manufacturer’s instructions (PeproTech^®, Princeton Business Park, Rocky Hill, NJ, USA) as previously performed [10 (link)]. The Abs₄₀₅ was measured using a Synergy HT microplate reader (Biotek) at room temperature (RT). Adherent cells were lysed with RIPA for protein quantification and Griess/ELISA results were normalized for total protein.