ELISA analyses were performed following the manufacturer's instructions for the different cytokines tested (IL-7, Peprotech, Neuilly sur Seine, France; CD40s, R&D Systems; ICOSL, Antibodies-online GmbH, Paris, France) on 100 μl of cell culture supernatant.
The blots were blocked with 2% blocking reagent (Blotting Grade Blocker, Bio-Rad) and incubated with either anti-fibronectin or anti-plasminogen antibodies (ABIN125555 or ABIN285631; antibodies-online GmbH, Aachen, Germany), diluted 1 1,000 in TBS.
Each serum sample was separated by 10% SDS-Polyacrylamid-gelelectrophoresis, transferred to PVDF membrane and detected with a primary antibody against PCG1-α (polyclonal IgG antibody coupled to HRP (Antibodies-online, GmbH) produced in goat, dilution 1 500 in TBST (is mixture of Tris-Buffered Saline and Tween 20) supplemented with 5% milk powder, incubation over night at 4°C).
Integrin expression of cultured BMMs was analyzed by using the following antibodies: Armenian hamster anti-rat CD29 (integrin β1, non-specific for activation status, PE-coupled, #562154; Becton Dickinson) and polyclonal rabbit anti-rat CD51/CD61 (integrin ανβ3, uncoupled, ABIN674784; antibodies-online GmbH, Aachen, Germany).
The sections were then subsequently rinsed in TBS/0.05% Tween buffer and incubated with primary antibodies (20 min at room temperature; RT) directed against CD34 (goat anti-rat, R&D Systems, Abington, UK) and VEGF-A (rabbit anti-rat, Antibodies-Online GmBH, Aachen, Germany).
Cell viability was evaluated by assessment of reduction of 3- 4,5-dimethylthiazol-2-yl 3- 4,5-dimethylthiazol-2-yl 3- 4,5-dimethylthiazol-2-yl 3- 4,5-dimethylthiazol-2-yl 3- 4,5-dimethylthiazol-2-yl 2nd lactate dehydrogenase (LDH) protein amount in cell culture medium by LDH ELISA kit (Antibodies-online, GmbH, Aachen, Germany).
The DA and questionnaires for evaluation were programmed in Unipark, an online survey tool (QuestBack GmbH, Cologne, Germany).
Tryptic peptides were analyzed by nanoLC-ESI-MS/MS using a nanoAcquity UPLC system (Waters GmbH) coupled online to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific).
Samples were analyzed using a nanoAcquity UPLC system (Waters GmbH) connected online to a LTQ-Orbitrap Velos Pro instrument (Thermo Fisher Scientific GmbH) as described by Ori et al (1999) Briefly, peptides were separated on a BEH300 C18 (75 μm × 250 mm, 1.7 μm) nanoAcquity UPLC column (Waters GmbH) using a stepwise 145 min gradient between 3 and 85% (v/v) ACN in 0.1% (v/v) FA.
The questionnaires were sent individually to the teachers via the Web-based "Survey-Online.com - Enuvo Gmbh - Zurich" − a Web application that enables distribution through emails containing the introductory letter, the URL of the actual questionnaire and a password to ensure security and that only one response would be received per respondent.
