Bio plex magpix system

The levels of inflammatory cytokines in the serum of rats in each group were detected by the Luminex liquid suspension chip (Wayen Biotechnologies, Shanghai, China). The Bio-Plex Pro Rat Cytokine 23-Plex Group I Panel System (Hercules, CA, United States) was used following the instructions of the manufacturer. Rat serum was incubated in 96-well plates embedded with microbeads for 1 h and then incubated with detection antibody for 30 min. Subsequently, streptavidin-phycoerythrin (PE) was added to each well for 10 min and values were read by using the Bio-Plex MAGPIX System (Bio-Plex) (Hercules, CA, United States).

To guarantee a fasting state, all study participants fasted for 8 h prior to blood sampling, and between 7:00 and 9:30 a.m., 5 ml of elbow venous blood were drawn from each participant. The serum samples were kept frozen in a −80 °C refrigerator after the blood samples were centrifuged at 3000 r/min for 15 min to separate the upper serum layer.
Serum neuropeptides were measured using the Bio-Plex MAGPIX System (Human Neuropeptide Magnetic Bead Panel, item number HNPMAG-35K, Bio-Rad), a suspension microbead microarray platform based on the Luminex liquid phase suspension chip. Serum neuropeptides were expressed as one trillionth of a gram per milliliter plasma (pg/ml).

The cytokines we measured included VEGF, inflammatory cytokines (IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, L-15, IL-17A, IL-18, IFN-g, and TNF-a), and chemokines (CXCL9, CXCL12, MIF, G-CSF, GM-CSF, M-CSF, IP-10, MCP-1, MIP-1a, MIP-1b, and RANTES) (Figure 1). The concentrations of vitreous cytokines were measured using Luminex multiplex technology (Bio-Rad Laboratories, Hercules, CA, United States) according to the manufacturer’s protocol with the assistance of Wayen Biotechnologies (Shanghai, Inc.). Briefly, 50 μl of vitreous sample or provided standard was added to 96-well micro-titer plate. After incubation at room temperature, diluted biotin antibody was then used to incubate with the samples, then added Streptavidin-PE for further incubation followed by wash buffer. Finally, the plate was read on the Bio-Plex MAGPIX System (Bio-Rad Laboratories).

A custom Luminex assay (R&D Systems, UK) for murine PlGF-2, VEGF, MMP9, IL-1β and IL-10 was used to quantify secreted protein levels in conditioned media collected from nTie2-iBMM and eTie2-iBMM cell cultures at days 3, 7, 14 and 21. The assay was carried out according to manufacturer’s instructions, and data captured using a Bio-Plex MAGPIX system (BioRad, UK).

Cell adhesion molecules (ICAM-1, VCAM-1, and P-selectin) were measured in plasma
by a commercial kit (Human Cardiovascular Disease Magnetic Bead Panel 2,
Millipore Sigma, USA) according to manufacturer's instructions. Quantification
of the magnetic beads was performed with a BioPlex MAGPIX system (Biorad, US)
and results were analyzed using Xponent software (Luminexcorp, USA). The
analyses of the concentration of cell adhesion molecules involved only eight
subjects because of technical problems with the plasma samples.

The Bio-Plex Pro Human Cytokine Grp I Panel including IL-1b, IL-2, IFN-g, TNF-a, and G-CSF was applied to detect cytokine secretion with the Bio-Plex MAGPIX System (Bio-Rad) according to the manufacturer’s instructions. Measurements were performed using the Bio-Plex MAGPIX Multiplex Reader. Plasma samples were collected and served as data for day 0. Supernatant fractions were collected at the indicated times for the measurement of cytokine secretion.

To verify the release of cytokines and growth factors into the biological media from the “Sticky Bone” preparations, an in vitro assessment was performed according to a previously described methodology [17 (link)]. The sticky bone and whole-blood samples (n = 3 per experimental group) were prepared as described and cultured for 7 days after their preparation in six-well culture plates (TPP, Merck KGaA Darmstadt, Germany) in the presence of 4 mL of DMEM medium (Dulbecco’s Modified Eagle’s Medium, GIBCO, Waltham, MA, USA), without antibiotics, in a humidified atmosphere at 37 °C and 5% CO₂. Aliquots of the conditioned medium were collected at the end of the incubation and stored in a freezer at −80 °C.
The concentration of cytokines and growth factors was detected in these samples through a multiparametric immunoassay based on XMap-labeled magnetic microbeads (LuminexCorp, Chicago, IL, USA). A commercial kit (27-plex panel, Biorad Inc., Hercules, CA, USA) was employed; it was capable of quantifying IL-1β, IL-10, IL-12 (p70), IL-13, IL-15, IL-10, IL-10, IL- IL-17, CCL11, FGF-b, CSF3, CSF2, IFN-γ, CXCL10, CCL2, CCL3, CCL-4, PDGF, CCL5, TNFα, and VEGF levels. Quantification of the magnetic beads was performed with a BioPlex MAGPIX system (Biorad Inc., Hercules, CA, USA). Results were analyzed using the Xponent v. 3.0 software (Luminexcorp, Chicago, IL, USA).