Sample diluent

Cytokine levels in rat brain protein lysate were measured using the Bio-Plex Pro Rat Cytokine Group I Panel 23-plex assay (BioRad, Cat # 12005641, Philadelphia, PA, USA) following the manufacturer’s instructions and as previously described [53 (link)]. Briefly, rat brain lysates were diluted 1:4 in Bio-Plex sample diluent (containing BSA to a final concentration of 0.5%). Standards were reconstituted and coupled beads were prepared following manufacturer’s instructions. Approximately 50 µL of coupled beads was added to each well in a 96-well plate, then washed prior to adding 50 µL of standard and samples (both assayed in duplicate) to the appropriate well in a 96-well plate. The plate was incubated and washed following manufacturers’ instructions, then read using a Bio-Plex 200 system. Innate immune protein concentrations were normalized to total protein as measured by Precision Red Advanced Protein Assay (Cytoskeleton, Inc.). Assay sensitivity and limit of detection (pg/mL) for each target: G-CSF (0.2), VEGF (0.3), IL-7/M-CSF (0.4), GM-CSF/GRO-KC (0.6), MIP-1α (0.7), IL-13 (0.9), IFN-γ/IL-1α/IL-4/ (1.0), IL-1β (2.0), IL-2/RANTES/TNF-α (3.0), IL-18/MCP-1 (4.0), IL-10 (5.0), IL-5 (6.0), IL-6 (10.0), and MIP-1α (12.0).

Concentrations of IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, Eotaxin, Basic FGF, G-CSF, GM-CSF, INF-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF were determined in plasma of BD patients and HC by the Bio-Plex Pro Human Cytokine Group I Panel, 27-Plex (Biorad) following the manufacturer's instruction. Plasma was diluted four-fold in Bio-Plex Sample Diluent as recommended. Data were obtained with the MAGPIX™ Multiplex Reader instrument and Bio-Plex^® Manager^TM software. Values extrapolated from the standard curve were considered not reliable and a concentration = 0.01 pg/mL was arbitrary assigned (Table S3 for the lower limits of detection).

Bio-Plex Pro Mouse Cytokine 23-plex assay (Bio-Rad, M60009RDPD) was used to compare the concentration of inflammatory cytokines and chemokines in hippocampal cytosolic fractions from ASIC2a +/+ and +/- pregnant mice. Samples were thawed and diluted 1:2 with Bio-Plex sample diluent and run following manufacturer’s directions. Standard curves were generated for each analyte and sample concentrations determined. All measured concentrations were normalized to protein concentration in each sample.

Cryopreserved PDA tissue samples were cut into multiple consecutive sections (20 µm) and then stained with cresyl-violet. Areas of tumor epithelium and stroma were highlighted using brightfield microscopy at 20× magnification. Laser microdissection was performed with the Leica Laser Microdissection V5.0.2.0 software. Sufficient protein concentrations for multiplex analysis could be obtained by the dissection of 30–50 × 106 mm². The tissue was lysed using the BioPlex™ Cell Lysis Kit (Bio-Plex Cell Lysis Kit, BioRad, Hercules, CA, USA; 171304011) according to the manufacturer´s instructions. Serum samples were thawed overnight at 4 °C and then diluted at 1:1 using Sample Diluent (BioRad, Hercules, CA, USA) prior to protein quantification. A two-laser array reader simultaneously quantified all proteins of interest. The concentrations were calculated using Bio-Plex Manager 4.1.1 and a 5-parameter logistic plot regression formula. The Bio-Plex Pro Human Cytokine Screening Panel 48-plex (BioRad, Hercules, CA, USA; 12007283), Bio-Plex Pro Human Cytokine ICAM-1 (BioRad, Hercules, CA, USA; 171B6009M) and Bio-Plex Pro Human Cytokine VCAM-1 (BioRad, Hercules, CA, USA; 171B6022M) were used for the quantification of immunological parameters.

First, serial sections (20 µm) of cryopreserved PDA tissue samples were stained with cresyl-violet. Then, Laser Capture Microdissection was performed with the Leica Laser Microdissection V5.0.2.0 software to separate the stromal compartment from tumor epithelium. The procedure was performed according to the protocol of the inventors (11 (link)). Both, the stromal tissue as well as the 3D bioprints were lysated using the BioPlex™ Cell Lysis Kit (Bio-Plex Cell Lysis Kit, BioRad, USA; 171304011) according to manufacturer´s instructions. Serum samples were thawed overnight at 4°C and diluted 1:1 prior to protein quantification (Sample Diluent, BioRad, USA). All proteins of interest were concurrently quantified using a two-laser array reader. Concentrations were calculated with Bio-Plex Manager 4.1.1 based on a 5-parameter logistic plot regression formula. Bio-Plex Pro Human Cytokine Screening Panel 48-plex (BioRad, USA; 12007283), Bio-Plex Pro Human Cytokine ICAM-1 (BioRad, USA; 171B6009M) and Bio-Plex Pro Human Cytokine VCAM-1 (BioRad, USA; 171B6022M) were utilized to quantify cytokine concentrations.