Mitotracker orange cmtmros

Mitochondria were stained with MitoTracker® Orange CMTMRos (Invitrogen, M7510) before fixation, in accordance with the manufacturer’s instruction. Cells were incubated for 30 min with the mitochondrial dye at final concentration of 190 nM in serum-free culture media, and then washed three times in serum-free culture media before fixation and immunofluorescence analysis.

Immunofluorescence microscopy was performed according to conventional procedures. Briefly, cells were fixed with methanol 100% for 10 minutes at −20 °C and then washed with PBS before nucleus staining with a dilution of Hoechst 33342 (1/5000 in PBS) or 4′,6′-diamidino-2-phenylindole (DAPI, 1/5000 in PBS) 10 minutes at ambient temperature. For staining mitochondria, cells were labeled during 30 minutes with the MitoTracker Orange CMTMRos (Invitrogen) (1/1000) then fixed and stained with Hoechst to visualize the nucleus. Microphotographs with the objective 20 and 40 were taken for analyses and count. Images were analyzed with the open source software Image J (freely available from the National Institute of Health, Bethesda, MD, USA at the address http://rsb.info.nih.gov/ij/).

Mitochondrion-selective probes, including MitoTracker^® Green FM (Invitrogen, Carlsbad, CA, USA) and MitoTracker^® Orange CMTMRos (Invitrogen, Carlsbad, CA, USA), were used to label the mitochondria of the NK cells. TD139 (TargetMol, Wellesley Hills, MA, USA) was dissolved in dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China) and used for the inhibition of Galectin-3 in vivo (15 μg/g body weight, once per 24 h for three times). A Galectin-3 mouse ELISA Kit (Invitrogen, Carlsbad, CA, USA) was used to determine the serum and liver homogenate levels of Galectin-3. A mouse IL-10 ELISA kit (Abcam, Cambridge, MA, USA) was used to determine the serum and liver homogenate levels of IL-10.

Freshly isolated human PBMC were surface stained for CD3/CD4/CD8/CD45RA/CD27. For FoxP3 intracellular expression, freshly isolated PBMC were stained with CD3, CD4, CD25 surface markers, and fixed, permeabilized according to eBioscence manufacture protocol and then stained intracellularly with anti-FoxP3 mAb or isotype control. Cytokines production in human cells was evaluated on freshly isolated PBMC after stimulation for 4 hours with PMA (0.05 ug/ml) and Ionomycin (1 ug/ml), of which the last 2 in the presence of Brefeldin A (5 ug/ml). Next, cells were surface stained, fixed and intracellularly stained for IL-2 and IFN-γ. Acquisition and analyses were performed using FACSCanto cytometer (BD Biosciences) and FlowJo Software (Tree Star). Mouse splenocytes were stained with directly conjugated mAb from BioLegend and BD Pharmingen (CD4/CD8/CD44/CD62L). Apoptotic cells were detected by Annexin V (BD Pharmingen) and TO-PRO-3 (Invitrogen) staining. Mitotracker Orange CMTMRos (Invitrogen) was used according to manufacturer instruction to measure mitochondrial membrane potential.