Bio plex protein array system

Serum and bronchoalveolar lavage fluid (BALF) samples were collected from the infected mice at indicated time points. For cytokine and chemokine measurements, serum and BALF samples were processed using the Bio-Plex Mouse Cytokine 23-Plex instrument (Bio-Rad Laboratories) following the manufacturer's instructions, and array analyses were performed using a Bio-Plex Protein Array system (Bio-Rad Laboratories).

Concentrations of VEGF-α, CCL5 (also known as regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL1 (also known as Gro/KC) in whey and serum samples were measured using a Milliplex Rat Cytokine/Chemokine Panel (RCYTO-80K, Millipore, Billerica, MA). Insulin concentrations in whey and serum samples were measured using a Milliplex Rat Bone Panel 1 (RBN1-31K, Millipore). These panels were processed using a Bio-Plex Protein Array System and the data were analyzed with Bio-Plex Manager 6.0 software (both from Bio-Rad Laboratories, Hercules, CA).

We retained one aliquot for further testing of selected growth factors (GFs), in particular, IL-10, IL-13, basic FGF, PDGF-bb, b-NGF, EGF, and TGF-α. Samples were evaluated by using commercially available multiplex bead-based sandwich immunoassay kits (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in the Bio-Plex Protein Array System (Bio-Rad Laboratories) as previously described [14 (link)]. This system consents a simultaneous quantitative analysis of multiple different factors in a single microliter well. Briefly, we used different sets of fluorescently dyed beads, loaded with capture monoclonal antibodies, specific for each tested cytokine. We measured and quantified the formation of different sandwich immune complexes on distinct bead sets. We discarded values with a coefficient of variation above 10% before performing data analysis with the Bio-Plex Manager software (version 6.0, Bio-Rad Laboratories). We considered only standard levels between 70% and 130% of the expected values. Eye drops used in the present experiments contained the following GFs (pg/mL), as reported in Table 1.
We treated the animals four times a day, administrating a drop of approximately 15 microliters per time, so meaning that each day, the treated eye received approximately:
EGF: 53.4 pg; TGF-α: 3.996 pg; PDGF-bb: 426.18 pg; FGF-basic: 4.65 pg; bNGF: 0.042 pg; IL10: 0.924 pg; and IL13: 15.762 pg.

Cytokine levels within lung tissue homogenates were analyzed using the Bio-Plex protein array system (Luminex-based technology; Bio-Rad Laboratories, Hercules, CA). Briefly, lung tissue was excised and homogenized in ice-cold sterile PBS (1 ml). An aliquot (50 μl) was taken to quantify the pulmonary fungal burden and an anti-protease buffer solution (1 ml) containing PBS, protease inhibitors, and 0.05% Triton X-100 was added to the homogenate. Samples were then clarified by centrifugation (3,500 rpm) for 10 min. Supernatants from pulmonary homogenates and ex vivo macrophages were assayed for the presence of interleukin IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17A, keratinocyte-derived cytokine or KC (CXCL1), monocyte chemoattractant protein 1 or MCP-1 (CCL2), macrophage inflammatory protein or MIP-α (CCL3), MIP-β (CCL4), regulated upon activation normally T-expressed and presumably secreted or RANTES (CCL5), Eotaxin (CCL11), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) according to the manufacturer’s instructions.

Cytokine levels in lung tissues were analyzed using the Bio-Plex protein array system (Bio-Rad Laboratories, Hercules, CA). Briefly, lung tissue was excised and homogenized in 2 ml of ice-cold PBS containing 1× Pierce protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL). After homogenization of the lung tissue, Triton X-100 was added to a final concentration of 0.05%, and the samples were clarified by centrifugation. Supernatant fractions from the pulmonary homogenates were then assayed using the Bio-Plex Pro Mouse Cytokine 23-Plex (Bio-Rad Laboratories) for the presence of interleukin 1α (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte monocyte colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ), chemokine (C-X-C motif) ligand 1 (CXCL1)/keratinocyte-derived chemokine (KC), chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), CCL3/macrophage inflammatory protein 1α (MIP-1α), CCL4/MIP-1β, CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES), and tumor necrosis factor alpha (TNF-α).

Cytokine levels within lung tissue homogenates were analyzed using the Bio-Plex protein array system (Luminex-based technology, Bio-Rad Laboratories, Hercules, CA). Briefly, lung tissue was excised and homogenized in ice-cold sterile PBS (1 ml). An aliquot (50 μl) was taken to quantify the pulmonary fungal burden and an anti-protease buffer solution (1 ml) containing PBS, protease inhibitors (inhibiting cysteine, serine, and other metalloproteinases) and 0.05% Triton X-100 was added to the homogenate. Samples were then clarified by centrifugation (3500 rpm) for 10 minutes. Supernatants from pulmonary homogenates were assayed for the presence of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12(p70), IFN-γ, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony stimulating factor (GM-CSF) according to the manufacturer’s instructions.