Human cytokine antibody array

A human cytokine antibody array (Abcam, UK) was used to compare the secretion of cytokines secreted by MSCs in 2D and 3D, under primed and unprimed conditions. Supernatants from 2D and 3D were concentrated and diluted (respectively) up to four times, so that results from the arrays were directly comparable.
A human MMP array was used to assess the differences in secretion of tissue remodelling proteins in 3D cultures. Supernatants from at least two independent experiments were pooled to reach 1 ml of supernatant required for the membrane array.
For both arrays, densitometry data were obtained using ImageJ software, and normalised against the untreated values; therefore, the priming effect could be easily observable in 2D and 3D cultures. A PANTHER over-representation test [27 (link)] including all the analysed cytokines was performed against a human protein database to detect the main biological processes related to the overexpressed proteins.

For screening, a Human Cytokine Antibody Array (ab133998, ABCAm, Cambridge, UK) was performed on L-PRF CM (collected after 96 h total) and EX of four different healthy donors at a protein concentration of 10 mg/mL according to the manufacturer’s instructions (Thermo Scientific, Erembodegem, Belgium). Protein concentrations of the samples were determined with a bicinchoninic acid assay (BCA, Thermo Fisher Scientific, Waltham, MA, USA) following the user manual. Relative pixel density was measured using ImageJ/Fiji software 1.53 (NIH, Bethesda, MD, USA) to compare relative protein levels between L-PRF EX and CM. This analysis was previously performed by our group [50 (link),83 (link)]. This study reinterpreted the data of these arrays focusing on the neurotrophic factors and other growth factors present in both L-PRF subfractions that may affect neuronal cell functions. In addition, an Enzyme-Linked Immunosorbent Assay (ELISA) (Raybiotech, Peachtree Corners, GA, USA) was performed to quantify and verify vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and platelet-derived growth factor (PDGF) protein levels in L-PRF EX and CM collected at 24, 48, 96, and 144 h to evaluate the release of these growth factors from the clot over time.

Cytokine levels in culture media were examined using a Human Cytokine Antibody Array (120 targets, Abcam, Cambridge, UK) according to the manufacturers’ instructions. Culture media were harvested from 80–90% confluent cultures of iMSCs derived from 201B7 or 409B2 iPSCs and BM-MSCs. Unused medium (10% FBS/DMEM) was used as a background control. Signals were detected using a ChemiDoc MP Imaging System (Bio-Rad). Data were analyzed with Image Lab 6.0 (Bio-Rad).

Macrophages (HLA-DR⁺CD14⁺CD206⁺CD169^-) or DC (HLA-DR⁺CD123^-CD11c⁺) were isolated from human fetal lung tissue by FACS (Fig S1D). Isolated macrophages were cultured in TexMACS medium (Miltenyi Biotec, 130-097-196) supplemented with 100 ng/ml human recombinant M-CSF (Peprotech, 300-25), while DC were cultured in ImmunoCult-ACF DC Medium (STEMCELL Technologies, 10986) supplemented with ImmunoCult DC Maturation Supplement (STEMCELL Technologies, 10989) for 7 days. On days 3, 5 and 7 of culture, supernatant was removed/stored and replaced with fresh medium. Supernatants were centrifuged at 1,000 g to remove debris. Cytokines secreted into the supernatant were analyzed using a Human Cytokine Antibody Array (abcam, ab133997), according to the manufacturer’s instructions.

NHLF or adCAFs were dissociated and resuspended in 3.96 U/ml Thrombin:EGM-2MV at a concentration of 3.32M/ml. Equal volumes of NHLF/adCAF suspension and 5 mg/ml Fibrinogen were mixed, and 100 μl was dispensed into the 24-well plate well surface. Fibrin was gelled at 37 °C for 15 min. Next, HUVECs were dissociated and resuspended in 3.96 U/ml Thrombin:EGM-2MV at a concentration of 50M/ml. Equal volumes of HUVEC suspension and 5 mg/ml Fibrinogen were mixed, and 20 μl droplets were dispensed onto 0.4 μm pore size polycarbonate membrane Transwell inserts (Corning cat. no. CLS3413) in a 24-well plate. Fibrin was polymerized at 37 °C for 15 min. Transwell inserts containing HUVECs were carefully placed into wells with NHLF/adCAF, and 600 μl EGM-2MV media was added to each well. Cultures were maintained for five days with serum-free media replacement on day 3. Supernatant was collected, spun at 15 000 RPM for 5 min at 4 °C, and stored at −20 °C until use. Supernatants were subjected to the Human Cytokine Antibody Array (Abcam cat. no. ab133997), according to the manufacturer's instructions.

WB was performed using conventional WB procedures. Primary antibodies were purchased from CST (Cell Signaling Technology, Danvers, MA, USA), including anti-HGF anti-phospho Met (#3133), anti-cMet (#8198), anti-Akt (#2920), anti-phospho-Akt (ser473, #4060), anti-pPDGFR (Tyr1009, #3124), and anti-phospho-Erk1/2 (Thr202/Tyr204, #4370).
Human cytokine antibody array (Abcam, Cambridge, USA, ab133998) was performed according to the manufacturer's instruction manual.
For MET activation and neutralization experiments, RKO cells were seeded in 24-well plates at a density of 40,000 cell per well in culture medium containing 10% FBS. One day before treatment, the wells were refreshed with medium containing 2% FBS. CAF CM diluted with culture medium (2% FBS) at 1:2 was added to the cells for 20 min at 37°C. Alternatively, the HGF neutralizing antibody was added to the diluted CAF CM and incubated for 1 h before treatment. The cells were lysed with a RIPA-containing phosphatase inhibitor cocktail for preparation of the WB samples.