Rat collagen type 1

Three-dimensional collagen type I was prepared using eight-parts rat collagen type I (Sigma) and one-part 10× DMEM and was neutralized to pH 7.0–7.5 by adding 0.5 parts 0.5N NaOH; 400 µl collagen I gel was then added immediately to each well of a 24-well plate. Endothelial cells (e.END4.1, e.ENDwt) were seeded onto the collagen type I gel base and were incubated at 37°C overnight. Splenic monocytes were isolated by depleting CD19, Ly6G, CD11c, CD3ε, Siglec-F, MHCII, and CD11c positive cells using magnetic beads (Miltenyi Biotec); 5 × 10⁵ cells were plated onto the confluent endothelial cells for 16 h at 37°C. The conditioned medium was collected for cytokines analyses, and remaining unattached cells were removed by 5× washing with DMEM; 400 µl Collagenase D (2 mg/ml, Sigma) was then added and the cultures digested at 37°C for 30 min. Cells were harvested and analyzed by flow cytometry for PECAM-1, Ly6C, MHCII, CD11b, and F4/80 expression. The same experiments were performed with splenocytes preincubated for 30 min in the presence of 25 μg/ml integrin α6 function blocking antibody (GoH3) or an isotype control. Experiments were performed three times using cells from two mice. Cytokines were measured in 5× concentrated conditioned media using Th1/Th2 10 Plex Flowcytomix Kit (eBioscience).

human collagen type III, human collagen type I, and rat collagen type I were purchased from Sigma. According to Sigma, human collagen type I and type III was purified from placenta, and rat collagen type I from tail tendon. The purchased collagens were solubilized in 20 mM acetic acid, pH 3, at 4°C and 2.4 mg/mL. Fresh rat collagen type I was prepared from a single rat tail tendon following a procedure published by Dr. Sergey Leikin’s group, and the precipitated collagen was solubilized in 20 mM acetic acid, pH 3, at 4°C and 3.0 mg/mL [46 (link)]. Collagen was mixed with 5X SDS sample loading buffer containing 60 mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 350 mM DTT, and 0.1% bromophenol blue, and was run on a 4–20% Precise gel or a 7.5% SDS PAGE gel and stained with coomassie blue. Bands of alpha chains were excised from the gels and submitted to The Rockefeller University for in-gel digestion and mass spectrometry analysis.

The THP-1 monocyte cell line obtained from the American Type Culture Collection (ATCC, Virginia, VA, USA) was grown at a density of 2 × 10⁶ cells in a 75 cm² flask in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% penicillin and streptomycin (PS, 100 U/mL and 100 µg/mL, Sigma-Aldrich), pyruvate (1 mM, Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), and 14.3 mM 2-mercaptoethanol (Sigma-Aldrich).
Immortalized human aortic endothelial cells TeloHAEC (ATCC^® CRL-4052^TM) were cultured in vascular cell basal medium (VBM, ATCC PCS-100-030) supplemented with vascular endothelial cell growth kit-VEGF (ATCC PCS-100-041: 5 ng/mL rhVEGF, 5 ng/mL rhEGF, 5 ng/mL rhFGF basic, 15 ng/mL rhIGF-1, 10 mM L-glutamine, 0.75 U/mL Heparin sulfate, 1 µg/mL Hydrocortisone, 50 µg/mL Ascorbic acid and 2% FBS) and 1% penicillin/streptomycin.
The immortalized human brain microvascular endothelial cell line (hCMEC/D3, SC0066, Merck, Branchburg, NJ, USA) was seeded on a 75 cm² flask precoated with 0.7 mg/mL rat collagen type I for 2 h (08-115, Sigma-Aldrich) and cultured in EndoGRO^TM-MV complete culture media (SCME004, Merck).
All cell lines were maintained in a humidified environment at 37 °C with 5% CO₂, and the medium was renewed every 2–3 days.