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Blood, Vol. 113, Issue 24, 6246-6257, June 11, 2009
Endothelial cell activation leads to neutrophil transmigration as supported by the sequential roles of ICAM-2, JAM-A, and PECAM-1
Blood Woodfin et al.
113: 6246
Supplemental materials for: Woodfin et al
Animals
Male (20–25g) wild-type (WT) mice were from Harlan-Olac (Bicester UK). ICAM-2 deficient (ICAM-2−∕−), JAM-A–deficient (JAM-A−∕−), and PECAM-1–deficient (PECAM-1−∕−) mice were used in this study. The JAM-A−∕− and ICAM-2−∕− mice were generated as previously described.12,16 PECAM-1−∕− mice were obtained as gift from Dr Tak Mak (Amgen Institute, Toronto, Canada).17 TNFα receptors p55 and p75 double knock out (TNFR−∕−) mice were purchased from Jackson Laboratories (Main, USA). Male (20–25g) WT mice on SJL and FVB/n background were purchased from Harlan (Bicester, UK). All genetically modified mice were on a C57BL/6 background. The experiments were carried out under the UK legislation for the protection of animals. Reagents
Recombinant murine IL-1β and TNFα were purchased from R&D Systems (Abingdon, Oxford, UK), Calcein-AM was purchased from Molecular Probes (Poortgebouw, The Netherlands). All other purchased reagents were from Sigma-Aldrich (Poole, Dorset, UK). The blocking anti–JAM-A mAb BV-11,18 and the blocking anti–JAM-C mAb H33 were generated as previously detailed.20 Anti–PECAM-1 mAb (Mec13.3), anti–ICAM-2 mAb (3C4), and anti–Mac-1 mAb (5C6) were from BD Biosciences (Cowley, Oxford, UK). Control IgG2b and IgG2a antibodies were from Serotec (Oxford, UK). The following antibodies were also used for immunofluorescence labeling for confocal imaging: Rat anti-mouse MRP-14 was a gift from Dr Nancy Hogg (Cancer Research UK, London). This mAb was conjugated to Alexa-Fluor-555 using a commercially available kit (Invitrogen, Paisley UK). Rabbit anti-mouse collagen IV (Abcam, UK), Allophycocyanin-conjugated anti–PECAM-1 (Mec13.3) from Cambridge Bioscience (Cambridge, UK), anti–rat-488, anti–rabbit-633, and anti–rabbit-546 Alexa-Fluor secondary antibodies (Invitrogen, Paisley, UK), anti-mouse α-smooth muscle actin (αSMA)-cy3 Sigma-Aldrich (Poole, Dorset, UK).
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