Bio plex 200 plate reader

3D microtissue samples were collected by centrifugation at 1000 rpm for 5 min at 4 °C and rinsed twice with PBS as described above for RNA isolation. Pelleted samples were lysed on ice in PBS containing 0.1% Triton X-100, 1 mM EDTA, and cOmplete™ Protease Inhibitor Cocktail (Millipore Sigma, USA) followed by centrifugation to remove cell debris. The total protein concentration of each sample (mg/mL) was determined using a Bio-Rad DC Protein Assay kit (Bio-Rad, USA). For Luminex (USA) bead assays, 3-plex (MMP1, MMP2, MMP3) and 4-plex (IL-1β, IL-6, HGF, TNF-α) magnetic bead kits, including reagents, detection antibodies, and standards, were assembled by mixing pre-coupled magnetic beads selected from Bio-Rad’s Pro Human Inflammation (Cat#171-AL001M) and Human Cytokine Screening (Cat# 12007283) multiplex panels. All protein samples were assayed in duplicate using kit protocols supplied by the manufacturer. Data collection was performed on a Bio-Rad BioPlex 200 plate reader employing BioPlex Manager software. Final analyte levels (pg/mL) were corrected for total protein in each sample and reported as picogram of analyte per milligram of total protein.

Plasma harvested from whole blood was screened on a MILLIPLEX^® Bovine Cytokine/Chemokine 15-plex kit (BCYT1- 33 K; EMD Millipore, Billerica, MA, USA) utilizing antibodies to bovine IFN-γ, interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, macrophage inflammatory protein (MIP)-1α, IL-36 receptor antagonist (Ra), IP-10, macrophage chemo-attractant protein (MCP)-1, MIP-1β, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF)-A according to the manufacturer’s instructions and as reported by Smith et al. [30 (link)]. Briefly, plasma samples were diluted 1:2 in assay buffer before adding 25 μL of standards, quality controls, and samples to the plate in duplicate, followed by 25 μL of magnetic beads. The plate was sealed, covered with foil, and incubated for 2 h on a plate shaker at room temperature (RT). The plate was washed three times, and 25 μL of detection antibody was added to each well. After incubating the plate for 1 h at RT, 25 μL of streptavidin–phycoerythrin (PE) was added per well. The plate was sealed, covered, and incubated for 30 min at RT. The plate underwent a final series of washes, and then, 150 μL of drive fluid was added. Marker concentrations were measured on a Bio-Plex 200^® plate reader (Bio-Rad, Hercules, CA, USA). Quality control values for each marker were consistently within the range stated by the manufacturer.

We adapted the Luminex assay developed by C. Fenwick and coll. to detect anti-HIV-1 gp140z Env-specific IgG [23 (link)]. The in-house produced HIV-1 Env Gp140z trimer was associated with MagPlex beads using the manufacture’s protocol (Bio-Rad, France). Activated beads were washed in PBS followed by the addition of 6.3 μg of protein antigen (4°C overnight under agitation). Beads were then washed with PBS, resuspended in blocking buffer and finally in 150μl of storage buffer. Beads were counted with an Auto-2000 cell counter (Nexcelom) and kept protected from light at 4°C. Luminex beads were diluted at 50,000 beads/mL in PBS and 50μl was added in a Bio-Plex Pro 96-well Flat Bottom Plates (Bio-Rad). Following two 0.05% tween PBS on a magnetic plate washer (Bio-Rad), 50 μl of individual serum samples diluted at 1/100 in PBS, were added. Binding was performed at RT for 30 min under agitation, before adding an anti-mouse IgG-PE secondary antibody (45min at 0.5 μg/mL, ThermoFisher). Beads resuspended in 80 μl of Sheath fluid (Bio-Rad) were agitated 5 min at 700 rpm on the plate shaker then read directly on a Bioplex-200 plate reader (Bio-Rad) with 50μl of acquisition volume and DD gate 5,000–25,000 settings. Median fluorescence intensities (MFI) were exported using Bioplex Manager 6.1 software.