Coulter ac t diff 2

Blood samples were processed within 24 h of being collected in EDTA tubes. PBMCs were isolated via gradient centrifugation at 1850 g on Ficoll Paque (GE Healthcare, Mississauga, Canada). PBMC purity was determined with a cell counter (Coulter Ac T diff 2, Beckman Coulter, Montreal, Canada). Cells were stored at −80 °C in Trizol (Invitrogen, Burlington, Canada) until RNA extraction. Aliquots of one to two micrograms of the RNA samples were utilized for qPCR. After DNAse digestion (DNase I amplification grade, Invitrogen), inverse transcription was performed with an inverse transcriptase (iScript cDNA Synthesis Kit, Biorad, Saint-Laurent, Canada) for 5 min at 25 °C, 30 min at 42 °C, and 5 min at 85 °C. Complementary DNA (cDNA) was generated in the presence of different forward and reverse primers for the genes of interest: SERT and DAT, or primers for the housekeeping gene β2-microglobulin (β2). All primers were obtained from IDT, and their sequences are depicted in Table 1. The threshold cycle (CT) values obtained for the SERT or DAT were subtracted by the corresponding CT values of β2 to obtain the ΔCT values of the SERT or DAT. Relative SERT mRNA expression was calculated with the aid of the 2^−ΔΔCT method [46 (link)], using the clinical groups as the targets, and the healthy control group as a reference.

Hematocrit, hemoglobin, and complete blood counts with a 3-part differential were analyzed via an automated hematology analyzer (Coulter® AC·T diff 2™, Beckman Coulter, Brea, CA). Plasma volume shifts were calculated using the formula established by Dill & Costill [35] (link). Plasma concentrations of TNF-α, STNFR1, and STNFR2 were measured via commercially available high-sensitivity enzyme-linked immunosorbent assay (ELISA; R&D Systems, Inc. Minneapolis, MN, USA), per manufacturer’s instructions, using an Epoch™ 2 Microplate Spectrophotometer (BioTek, Winooski, VT, USA). To eliminate interassay variance, all samples for each assay were thawed once and analyzed in the same assay run by a single technician. All samples were analyzed in duplicate with a mean intra-assay variance of 4.46% for TNF-α, 2.79% for STNFR1, and 4.21% for STNFR2.