Alexa fluor 488 conjugated anti rabbit antibody

Cells were fixed with 4% (w/v) paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then stained with anti-SLC25A11 polyclonal antibody, Mitotracker (M22425, Invitrogen Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 488-conjugated anti-rabbit antibody (A11008, Invitrogen Life Technologies, Carlsbad, CA, USA). Live cell imaging was taken by LSM510 Laser Scanning Microscope and Axio Observer Z1 (Carl Zeiss, Oberkochen, Germany). The relative intensity was normalized by the arithmetic mean intensity (from Zen software 2.6 blue edition).

Expression of VCAM-1 on the surface of HUVECs was evaluated by incubating 2 × 10⁵ HUVECs in EGM in the absence or presence of 20 ng ml⁻¹ human TNFα (hTNFα Millipore, Billerica, MA, USA) for 24 h. After harvesting and washing with phosphate-buffered saline solution (PBS), the cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature. After blocking with PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature, the cells were incubated first with mouse anti-VCAM-1 monoclonal antibody (1 μg ml⁻¹; Abcam, Cambridge, UK) for 1 h at 37 °C, and then with Alexa Fluor 488-conjugated anti-mouse antibody (1:1000; Invitrogen) for 1 h at 37 °C. The effects of anti-VCAM-1-D6 IgG on endothelial cell activation were evaluated by incubating 2 × 10⁵ HUVECs in the absence or presence of 20 ng ml⁻¹ hTNFα (Millipore), 20 μg ml⁻¹ control IgG or anti-VCAM-1-D6 IgG for 24 h. After blocking with PBS containing 1% BSA for 1 h at room temperature, the cells were incubated first with rabbit anti-ICAM-1 monoclonal antibody (1:500; Abcam) for 1 h at 37 °C, and then with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:1000; Invitrogen) for 1 h at 37 °C. Samples were analyzed by flow cytometry using a FACSCalibur system (BD Biosciences, San Jose, CA, USA) with the aid of FlowJo software (TreeStar, Ashland, OR, USA).