Fitc conjugated rat anti mouse f4 80

Single cells were isolated from both normal and diseased kidneys using enzyme-digestion and analyzed by flow cytometry as previously described.⁴⁷ After permeabilization, cells were incubated with FITC-conjugated rat anti-mouse F4/80 (eBioscience) or rat anti-mouse CD11b (Serotec) followed by Cy5-conjugated goat anti-rat IgG (Millpore), PE-conjugated anti-α-SMA (R & D, Minneapolis, MN, USA), FITC-labeled rat anti-mouse CD206 (Serotec) or rabbit anti-CX3CR1(Prosci Inc). Collagen I was stained with rabbit anti-mouse collagen I (Millpore) followed by the Cy5 labeled goat anti-rabbit Ig (Invitrogen). Cells incubated with isotype-matched irrelevant control antibodies and unstained cells were used as negative controls. Cells were detected by a FACS Calibur flow cytometer (BD Biosciences) and analyzed using Cellquest software.

U937 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin. For myeloid differentiation of U937 cells, 1x10⁶ cells were induced once with 10nM PMA (phorbol 12-myristate 13-acetate). For differentiation of ABR siRNA transfected U937 cells, 1nM PMA was used. Mouse bone marrow cells were obtained from tibiae and femora from C57Bl/6 wild-type mice and were seeded at 1x10⁶/mL in Dulbecco’s modified Eagle’s medium (DMEM) complemented with 10% FBS, 1% penicillin-streptomycin and dexamethasone (10^–8 M). Differentiation of granulocytic and monocytic lineages were induced with G-CSF (1 ng/ml) or M-CSF (20 ng/ml), respectively. Treatment of the cells was done daily during the experimental process. Cell differentiation was assessed by flow cytometry analysis using PE-conjugated mouse anti-human CD11b antibody (BD Biosciences), FITC-conjugated rat anti-mouse F4/80 and FITC-conjugated rat anti-mouse Gr-1 (eBioscience). Apoptosis was measured with an Annexin V Apoptosis Detection Kit I (BD Bioscience) according to the manufacturer’s instructions. For Rac inhibition, NSC23766 (Selleckbiochem) was added daily to the cell culture medium.

Cells in BAL were stained with FITC-conjugated rat anti-mouse F4/80 (eBioscience), PE-conjugated rat anti-mouse Siglec-F (BD Biosciences, CA, USA), APC-conjugated hamster anti-mouse CD11c (eBioscience) and APC-conjugated rat anti-mouse Ly6G (BioLegend, CA, USA) antibodies. For phenotyping, stained cells were analyzed by FACSCanto II (BD Biosciences). Stained cells were sorted by FACSAria (BD Biosciences) through the service provided by the Flow Cytometric Analyzing and Sorting Core (the First Core Laboratory, National Taiwan University College of Medicine).