Heparin

Total Hg levels were measured in blood, hair and urine samples. Blood samples were collected in heparin or EDTA tubes (BD) by venepuncture, and were stored on ice until analysis. Approximately 20 strands of hair 1 cm in length (about 10 mg), were gathered and cut from the occipital region of the scalp. Each sample was placed in plastic bags and maintained at room temperature. Urine samples were collected in 50 mL sterile polyethylene tubes, and were kept at 4 °C. Chemical analyses were carried on using Cold Vapor-Atomic Absorption Spectrophotometry (CV-AAS), using certified reference materials for quality control (IAEA-085, International Atomic Energy Agency, Vienna, Austria; and SRM 3668 and 955c, National Institute of Standards and Technology, Bethesda, MD). Results are given either as μg/L or as μg/g of dry weight.

Growth factor reduced Matrigel (BD #356231) was mixed with 20 U/mL Heparin, 400 ng/mL recombinant mouse bFGF (Shenandoah), and 400 ng/mL recombinant mouse VEGF 165 (Shenandoah). WT and SlugKO male and female mice at 20–24 weeks of age were used, consistent with the mice used for the subcutaneous tumor model. A 400-μL bolus of Matrigel mix was injected subcutaneously on the ventral lower abdominal area of the mice, close to the mid-line. For some experiments, mice received unilateral injection of Matrigel containing isolated wild-type mouse fibroblasts³⁴ (200,000 cells per plug) and acellular Matrigel injected into the contralateral control side. In all experiments, Matrigel plugs were harvested 7 days post-injection, and fixed in 10% formalin overnight at room temperature. The plugs were then paraffin embedded and sectioned for IHC analysis. Each experiment was repeated three times with at least 5 mice per genotype each time.

Blood samples were collected from eight healthy volunteers (22–40 years old, with a BMI between 20–25, all female) after an overnight fast. For each subject, serum and plasma samples were collected into six different tubes that included plastic tubes with no additives (for serum), and acid citrate dextrose plasma (ACD), sodium citrate (Citrate), ethylenediaminetetraacetic acid (EDTA), sodium fluoride (Fluoride), and sodium Heparin (Heparin) (for plasma) (BD Biosciences, San Jose CA). Each plasma and serum sample was processed as designated by the manufacturer’s specification. Briefly, for plasma, collection tubes were inverted eight times followed by centrifugation at ≤1,300 RCF for 10 min at 20°C. Serum tubes were gently inverted five times followed by a 45–60 min resting period at room temperature to obtain complete coagulation before performing the same centrifugation process as for plasma. Each serum/plasma sample was aliquoted into three 1.0 ml fractions and stored at -80°C until metabolomics analysis. All volunteers gave their written informed consent prior to participation in the study. The study was approved by the institutional review board at the University of California, Davis.

PBMCs were isolated from whole blood anticoagulated with heparin (132 Units per 8 ml blood) (BD Biosciences, Oxford, UK) using standard methods. Of note is that the material used for density gradient centrifugation was adjusted dependent on the macaque species, with a Ficoll Histopaque gradient (GE Healthcare, USA) used with rhesus macaque blood and a Percoll gradient (GE Healthcare) used with cynomolgus macaques. Mononuclear cells (MNC) were isolated from spleen and lung tissue samples using an OctoMACS tissue dissociation device (Miltenyi Biotec). Lung tissue samples were dissected into approximately 5mm³ pieces and incubated for 1 h in a solution of 772.8 U/ml collagenase + 426 U/ml DNase (both from Sigma) diluted in Earle’s balanced salt solution supplemented with 200 mg/ml Calcium Chloride (Gibco, Life Technologies, Renfrew, UK), at 37 °C with continual gentle mixing of the tube. The homogenised solution was passed through a 70 µm cell filter (BD Biosciences) and the mononuclear cells separated by Ficoll Histopaque density gradient centrifugation. PBMCs and MNC isolated from tissues were stored at −180 °C until resuscitated for analysis.

The isolation of CMVECs was performed according to a published protocol [32 (link)]. Briefly, C57BL/6J mice were sacrificed, and the hearts were dissected into ≈1 mm³ pieces. The heart samples were then digested by the addition of 0.1% collagenase II (Sigma) until the tissue blocks disappeared (approximately 30 min), followed by 0.25% trypsin-EDTA (Sigma) for 10 min at 37°C. Subsequently, the CMVECs were collected by filtrating and centrifugation (1000 xg for 10 min), resuspended in 20% FBS (Gibco)-M199 medium (HyClone) containing 50 μg/ml heparin and 75 μg/ml endothelial cell growth supplement (BD Biosciences), and plated in a culture flask. After 48 h, the CMVECs were cultured in a complete medium containing 20% FBS. Microscopy and flow cytometry (FCM) were adopted to identify CMVECs. Cells were incubated with the following fluorochrome-conjugated primary antibodies: anti-CD31-FITC, anti-CD34-FITC, and anti-vWF-FITC (BioLegend). CMVECs between 3 and 5 passages were used for subsequent experiments. These CMVECs were exposed to 200 μM H₂O₂ for 3 h to establish the oxidative stress conditions for subsequent experiments [33 (link)].

Matrigel plug angiogenesis assay was performed as described previously [15 (link)]. Briefly, a mixture of BD Matrigel matrix (0.5 mL) and heparin (50 unit/mL; BD Bioscience) was subcutaneously injected into C57BL/6 mice (aged 6–7 weeks) with VEGF (50 ng/mL) and MSSV (125 and 250 µg/mL). After 7 days, mice were euthanized and the Matrigel plugs were removed. Vascularization was determined by measuring the hemoglobin content of the Matrigel plugs using the Drabkin method (Drabkin reagent kit 525, Sigma-Aldrich, Louis, MO, USA) as well as the infiltrated endothelial cell count in the Matrigel plugs via CD31 antibody staining. All animal experiments were performed with the approval of the Animal Care and Use Committee of Chungbuk National University.

¹¹¹In-cNGR, ¹¹¹In-coNGR, or ¹¹¹In-co(NGR)₄ was added to 1 × 10 mL human blood in heparin (BD Biosciences, Vianen, The Netherlands). Blood stability was tested at 0, 10, 30, and 50 min. For each time point imaging agents were separated from blood cells and proteins by adding 0.5 mL MeCN (VWR International BV, Breda, The Netherlands) to 0.5 mL blood sample followed by centrifugation (2,000 ×g, 3 min). Supernatant samples were analyzed by HPLC using the above-mentioned method.