2.10. Measurement of Adhesion Molecules and Hsp70 by Flow Cytometry

HMVEC, exposed to laminar shear stress or cultured under static conditions, were irradiated and stimulated with TNF-α. To evaluate the influence of Nrf2 activation on the protein expression of adhesion molecules, cells were treated with the Nrf2 activator AI-1 or DMSO as a control. After 24 h, cells were detached with citric saline buffer (0.135 M KCl, 0.015 M sodium citrate) and incubated with fluorochrome-conjugated cell surface antibodies specific for adhesion molecules (ICAM-1-APC, clone HA58, 1:10, #559771, BD; VCAM-1-PE, clone IE10, 1:10, #FAB5649P, Bio-Techne GmbH, Wiesbaden, Germany; E-Selectin-FITC, clone BBIG-E5, 1:10, #BBA21, Bio-Techne GmbH) for 30 min. After washing, cells were fixed and permeabilized (FIX&PERM^® Cell Fixation and Permeabilization Kit (Nordic-MUbio, Susteren, The Netherlands), and incubated with a PerCP-conjugated antibody directed against heat shock protein 70 (Hsp70; clone EP1007Y, 1:80, #ab223390, Abcam) for 30 min, followed by a washing step. Flow cytometry was applied (CytoFlex S, Beckman Coulter GmbH, Krefeld, Germany) and the mean fluorescence intensity (MFI) of each fluorochrome was calculated using CytExpert Software 2.4 (Beckman Coulter GmbH).