Cytation imaging microplate reader

Solvents of analytical-grade reagents and other consumables were obtained from Fisher Scientific Ltd. (Dublin, Ireland). For blood sampling, both 20G safety needles and evacuated sodium citrate S-monovettes were obtained from Sarstedt Ltd. (Wexford, Ireland), while standard BSA, PAF, thrombin and egg phospholipids were purchased from Sigma Aldrich (Wicklow, Ireland). All the other consumables related to platelet aggregation assays were purchased from Labmedics LLP (Abingdon on Thames, UK). An Eppendorf 5702R centrifuge (Eppendorf Ltd., Stevenage, UK) was utilized for all centrifugations of blood samples, while all platelet aggregation bioassays were performed on a Chronolog-490 two-channel turbidimetric platelet aggregometer (Havertown, PA, USA), coupled to the accompanying AGGRO/LINK software package. Spectrophotometric analysis and absorbance measurements for the evaluation of total phenolic content (TPC), total antioxidant activity (TAA) and total carotenoid content (TCC) were carried out using a Cytation imaging microplate reader (BioTek, Winooski, VT, USA).

The TPC of mushroom extracts was measured using the Folin-Ciocalteu reagent as described previously [23 (link)], with minor modifications. Thus, 25 µL of each standard or mushroom extract was mixed with 125 µL of a 10-fold diluted Folin–Ciocalteu reagent in microplate wells. Acidified ethanol (50 µL, 0.7% v/v) or sodium phosphate buffer (50 µL, 50 mM, pH 7.5) was added to analyze the lipophilic or hydrophilic phenolic content in the extracts, respectively. The resultant mixtures were incubated in darkness for 120 min, and the absorbances measured at 755 nm on a Cytation imaging microplate reader (BioTek, Winooski, VT, USA). The TPC of the mushroom extracts were calculated by summation of the hydrophilic phenolic content (HPC) and lipophilic phenolic content (LPC) and the concentration measured based on quercetin standard curve. The results were expressed as µmol quercetin equivalent/g extract (DW).

The TAA of the mushroom extracts was measured colorimetrically using the ferric reducing antioxidant power (FRAP) method [22 ], with minor modifications. Briefly, 20 µL of each standard or sample was reacted with 180 µL of freshly made FRAP solution, and the resultant mixture incubated in the dark for 120 min. Absorbance measurements were recorded at 593 nm on a Cytation imaging microplate reader (BioTek, Winooski, VT, USA). The TAA extracts’ values were determined by summing the lipophilic (LAA) and hydrophilic (HAA) antioxidant activity values based on a Trolox standard curve using the following concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5 mM). The results for the Chaga extracts were expressed in µmol Trolox equivalent (TE)/g extract (dry weight: DW).