Heparin

Human kidney microvascular endothelial cells (HKMECs) were purified from fetal kidneys after voluntary pregnancy interruptions between 100 and 135 days postconception. Informed consents for the use of fetal tissues were obtained from patients. We then randomly chose 9 different donor HKMECs, thawed and plated half a million cells in T25 flasks coated with 0.2% gelatin and maintained in EBM-2 basal medium containing 1% antibiotic-antimycotic (Life Technologies), 10% FBS, 100 μg/mL ECGS, 50 μg/mL Heparin, and 20 ng/mL VEGF (R&D), for 48 h till confluency. At 48 h, we purified genomic DNA from the HKMECs and cell supernatants were collected. We successfully genotyped cells from 8 donors.

C2C12 myoblasts were seeded and cultured as outlined above. Cultures were stimulated with 10 μg/ml heparin (Sigma, St. Louis, MO, USA) or heparin and 1 μg/ml Klotho (R&D Systems, Minneapolis, MN, USA) in a growth medium at 24‐ and 48‐h post‐plating. Following 48 h of stimulation, cells were collected in Trizol reagent for RNA isolation.

96-well plates (Nunc) were coated with human sCD40L (Biozol, Germany) or human fibrinogen, ICAM-1, heparin, RAGE, vitronectin, JAM-C, or NIF (10 μg/ml, all from R&D Systems, USA) and incubated with CHO cells expressing constitutively activated, human Mac-1 (Mac-1 del) or CHO cells expressing the naïve, non-activated Mac-1 (Mac-1 WT)^{43 (link)}. As controls, CHO cells expressing no integrin (CHO) were used. Cells were pre-incubated with blocking antibodies (10 μg/ml) for 15 min and allowed to adhere for 50 min. Adhering cells were counted after repeated washing with PBS. Alternatively, we tested the adhesion of primary mouse peritoneal macrophages to mouse CD40L (R&D Systems, USA). For dynamic adhesion assays, mouse endothelial cell cultures (HUVECs) were grown to confluency in 35 mm cell culture dishes as previously described^{41 (link)}, stimulated with TNFα overnight, and placed in a parallel flow chamber system (Glycotech). The number of adhering cells was quantified at the indicated shear rate in the presence of the indicated antibodies (10 μg/ml).