Human high sensitivity t cell magnetic bead panel

TRD patients provided blood samples at baseline, day 13, and day 26, and HCs provided only a single blood sample. Blood samples were collected into EDTA tubes between 8:00 and 10:00 AM after an overnight fast. The tubes were immediately stored at + 4 °C and then centrifuged (3000 rpm/min at + 4 °C) for 10 min within 1 h. The plasma was obtained and stored at − 80 °C.
Plasma inflammatory cytokine levels were detected using the Human High Sensitivity T Cell Magnetic Bead Panel (Millipore, Billerica, MA, USA, HSTCMAG-28SK) performed with a Luminex 200 multiplex immunoassay system based on the manufacturer’s instructions. Nineteen cytokines, including interferon-inducible T cell alpha chemoattractant (ITAC), granulocyte-macrophage colony stimulating factor (GM-CSF), fractalkine, interferon (IFN)-γ, IL-10, macrophage inflammatory protein (MIP)-3a, IL-12P70, IL-13, IL-17A, IL-1β, IL-2, IL-4, IL-23, IL-5, IL-6, IL-7, IL-8, MIP-1β, and TNF-α, were quantified twice. The detailed detection process and interassay coefficients of variability have been described in our previous studies [27 (link)].

Blood samples were centrifuged at 3000 revolutions per minute at 4 °C for 10 min and frozen at −80 °C before further processing. The concentrations of 19 cytokines, including interferon-inducible T cell alpha chemoattractant (ITAC), GM-CSF, fractalkine, IFN-γ, IL-10, macrophage inflammatory protein (MIP)-3α, IL-12p70, IL-13, IL-17A, IL-1β, IL-2, IL-4, IL-23, IL-5, IL-6, IL-7, IL-8, MIP-1β, and TNF-α, were detected in plasma using a human high-sensitivity T cell magnetic bead panel (Millipore, Billerica, MA, USA, HSTCMAG-28SK) with a Luminex 200 multiplex immunoassay system based on the manufacturer’s instructions. Milliplex Analyst 5.1 software (EMD Millipore, Billerica, MA) was used with a cubic curve fitting to calculate the cytokine concentrations of the samples. All samples were run in duplicate, and the interassay coefficients of variability were <20%.

The concentrations of selected cytokines in the plasma samples derived from 51 IR subjects and 24 INR subjects were detected using a human high sensitivity T-cell magnetic bead panel (Millipore) according to the manufacturer’s instructions.
The samples underwent incubation with magnetic beads, detection antibodies and Luminex200 detection. After the samples and standards tested in this experiment were detected by Luminex 200 detector, the fluorescence obtained was automatically detected and optimized by software to form the raw data. According to the fluorescence detection value obtained from the standard, the standard curve and its equation are obtained by fitting the standard curve with multi-parameter mode, and the concentration unit is pg/ml. The original fluorescence detected by each sample was substituted into the standard curve formula to calculate the sample concentration, used for comparison between samples.