Annexin 5 fitc pi

Cell death was measured through the Annexin V-FITC/PI (fluorescein isothiocyanate/propidium iodide) Apoptosis Detection Kit (R&D Systems, USA), according to the direction from the manufacturer. The fluorescence intensity of Annexin V-FITC/PI was detected through flow cytometry (BD Bioscience).
For quantifying the renal macrophage polarization in mouse kidneys, kidney tissues were minced into 1 mm³ fragments and then digested in RPMI 1640 buffer containing 100 U/mL DNase I and 2 mg/mL collagenase type D for 60 min at 37°C and then passed through a 70 μm mesh to get single-cell suspension. Red blood cell lysis buffer (Sigma, USA) was used for lysing the red blood cells in the suspension. Mps were centrifuged and then resuspended in FACS buffer on ice. Incubated with 2.5 μg/mL Fc-blocking solution, Mps were resuspended in FACS buffer. Then, 10⁶ cells were stained with 3 fluorochrome-labeled antibodies: F4/80 (eBioscience)-PE, CD11c-FITC, and CD206-FITC. Finally, Mps were detected immediately on a FACS Canto II cytometer with DIVA software (Becton Dickinson). The data were analyzed by Cyflogic V.1.2.1 software.

RPMI 1640 cell culture medium, PBS, and penicillin/streptomycin were obtained from Hyclone (Thermo Scientific, Waltham, Massachusetts, USA). Fetal bovine serum (FBS) was obtained from GIBCO (California, USA). ALA hydrochloride powder was obtained from Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co, Ltd (Shanghai, China). The following chemicals and commercially available assay kits were used: apoptosis detection kit Annexin-V-FITC/PI (R&D Systems, Minneapolis, MN), rabbit anti-Mouse CD80-FITC, rabbit anti-Mouse CD86-FITC, rabbit anti-Mouse MHC-II-PE, rat IgG2a K Isotype Control FITC, Armenian Hamster IgG Isotype Control PE (eBioscience, USA), Hoechst 33342/PI kit (Beyotime Institute of Biotechnology, China), Mouse IFN-γ, IL-12 and IL-10 ELISA Kit (R&D Systems), and MTT assay Kit (Sigma-Aldrich, St Louis, MO, USA).

The analysis of DCs on flow cytometer (Coulter, XL-MCL, Krefeld Germany) or confocal microscope (Zeiss LSM 510/Axiovert 200 M, Jena, Germany) was performed upon labeling the cells with primary antibodies (Abs). The following Abs (clones) and reagents were used for the immunocytochemistry and flow cytometry: IgG1a negative control–biotin (MCA928), anti-CD1a-phycoerythrin (PE) (NA1/34HLK), IgG1 negative control–PE (MCA928PE), anti-CD14-Fluorescein isothiocyanate (FITC) (TUK4), anti-CD86–FITC (BU63), anti-CD3-FITC (UCHT1), IgG1 negative control–FITC (MCA928F) (all from Serotec, Oxford, UK), anti-CD45-PECy5 (HI30), anti-HLA-DR-biotin (LN3), IgG1a negative control-PECy5 (P.3.6.2.8.1) (all from eBioscience), streptavidin-Alexa 488, anti-CD83-Alexa 488 (all from Biolegend). The expression of HLA-DR, CD86 and CD83 was analyzed within CD45⁺ DC population. Necrosis of DCs cultivated with GNPs (5–200 µg/ml) was measured after 48 h-cultures by staining the cells with propidium iodide (PI, 10 µg/ml Sigma) in phosphate buffer saline (PBS). Apoptosis was determined after 48 h by staining the cells with PI in hypotonic citric/Triton-X buffer, or after 24 h by Annexin-V-FITC/PI (R&D) labeling.