Synergy ht multi detection reader

MRC5 human male foetal lung fibroblasts, SV40 transformed, and MDA-MB-231, human Caucasian female breast adenocarcinoma cells were sourced from the European Collection of Cell Cultures (ECACC) and maintained in Minimal Essential Medium with Glutamax and Earl's salts (MEM, Gibco), supplemented with 10% (v/v) foetal bovine serum (FBS, Pan Biotech) at 37° C in a humidified atmosphere with 5% CO₂. Cell passage was performed when cells were 70–80% confluent. Cells were first washed with Dubecco's Phosphate Buffered Saline without calcium chloride or magnesium chloride (DPBS, Sigma Aldrich) prior to the addition of cell dissociation agent, TripLE Select using 1 mLcm⁻² (ThermoFisher Scientific). Cell viability was measured with the CellTiter-Blue reagent (Promega) per the manufacturer's instructions. Cells were plated in clear-bottomed 96-well plates at a density of 5000 cells per well. The inhibitors were added the following day, and cell viability was measured 24 hours later using the Synergy HT Multi-Detection Reader (BioTek). Relative cell viability at a given inhibitor concentration was determined by comparing the fluorescence to that of DMSO treated cells.

After indicated drug treatment, cells (2 × 10⁴/ml) in 96-well plates were incubated with 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 37 °C for 60 min. The culture medium was removed, and then the formazan granules generated by live cells were dissolved in DMSO. The net absorbance (OD₅₅₀ - OD₆₃₀) indicating the enzymatic activity of mitochondria and cell viability was measured. In some experiments, cytotoxicity was assessed by measuring the release from damaged cells of the cytosolic enzyme lactic dehydrogenase (LDH). After treating cells with the indicated agents, LDH activity present in the culture medium was determined using a LDH diagnostic kit (Promega, Heidelberg, Germany). The absorbance values were read at OD₄₉₀ on a Synergy HT Multi-Detection Reader (Bio-Tek Instruments, Winooski, VT, USA). Data were expressed as the percentages of total cellular amount of LDH that was determined by lysing cells with lysis buffer (0.9 % Triton X-100).

Total RNA was extracted from 15 mg of liver (n = 9 per treatment), using NZYol (Nyztech, Lisbon, Portugal) following the manufacturer’s instructions, with some modifications, as previously described by Ferreira et al. (2020) (link). RNA quantity and purity (A260/A280 ratios) were evaluated by spectrophotometry, using a Take 3 micro-volume plate on a Synergy™ HT Multi-Detection Reader and Gen5™ software (BioTek Instruments, Winooski, VT, United States). RNA integrity was assessed in 1% TAE (w/v) agarose electrophoresis gel stained with GelRed™ (Biotium, Hayward, United States). Subsequently, cDNA was synthesized from 1 μg of total RNA using a NZY First-Strand cDNA Synthesis Kit (Nyztech, Lisbon, Portugal), following the standard protocol.

According to the manufacturer's protocols, direct differentiation of human UC‐MSCs toward adipocytes, osteoblasts, and chondrocytes was initiated with a Human MesenCult Adipogenic Differentiation Kit (05412, STEMCELL Technologies Inc., Canada), Human MesenCult Osteogenic Differentiation Kit (05465, STEMCELL Technologies Inc., Canada), and MesenCult‐ACF Chondrogenic Differentiation Kit (05455, STEMCELL Technologies Inc., Canada), respectively. Two to four weeks later, the cells were either fixed for Oil Red (O1391, Sigma–Aldrich, USA) staining and immunostaining or exacted by using RNA isolate Total RNA Extraction Reagent (R401‐01, Vazyme, China) for RNAs. To calculate the differentiation efficiency of adipocytes, photos of three fields at random were taken with a 10× objective lens with NIS‐Elements F software by a microscope (Nikon Ts2‐FL, Japan) for the UC‐MSCs after Oil Red O staining. Then the cells were counted with the ImageJ 1.52v software for further analysis. For quantification of lipids from Oil Red‐stained cells, 100% isopropanol was used to extract Oil Red for 5 min with gentle rocking. The absorbance at 492 nm was read by a Synergy HT Multi Detection Reader (BioTek Instruments, Winooski, VT) for the extracted liquid.

Our procedure was adapted from that described previously [35 (link)]. The larval fish food was fluorescently labeled paramecia prepared using the lipophilic tracer 4-(4-(Didecylamino)styryl)-N-Dethylpyridinium iodide (4-Di-10-ASP; Invitrogen, Carlsbad, CA, USA). We conducted feeding of 7 dpf zebrafish in 6-well plates to allow for free swimming. At 1.5 h after feeding, the larvae were anesthetized. After two washes to remove residual paramecia, groups of five larvae were transferred into a 96-well round-bottom black plate in an anesthetic solution. The intra-abdominal fluorescent signal was measured using the Synergy™ HT Multi-Detection Reader (BioTek Instruments, Winooski, VT) in fluorescence area scan mode 11 × 11 multipoint/well, 0.1 s/point using a fluorescein filter set (excitation wavelength, 485 nm; emission wavelength, 590 nm).