Armenian hamster igg isotype control fitc

Spleen and kidney specimens were obtained from the mice after euthanasia, homogenized, and filtered through 70 μm cell strainers (BD Falcon) for single-cell suspension preparation. Red blood cells were lysed three times with ammonium chloride-potassium lysing buffer (Quality Biological, Inc.) for 5 min. The obtained cells were counted with a cell counter (Countstar) and adjusted to 1∗105/ml. The cells were transferred into flow cytometry tubes and stained with anti-mouse gamma delta TCR FITC or Armenian hamster IgG isotype control FITC (eBioscience) for 30 min. This was followed by staining with anti-mouse CD3e PE-Cyanine5.5 (eBioscience), PE/cy7 anti-mouse CD28 or PE/cy7 mouse IgG2b, Κ isotype ctrl (BioLegend), and APC anti-mouse CD195 (CCR5) or APC Rat IgG2a, Κ isotype ctrl (BioLegend), and Brilliant Violet 421 anti-mouse CD192 (CCR2) antibody or Brilliant Violet 421 Rat IgG2b, Κisotype ctrl (BioLegend) for 30 min at 4°C in the dark. Flow cytometry was performed on a FACSCanto II (BD); data analysis was carried out with the FlowJo software (Tree Star).

Dulbecco's Modified Eagle Medium (DMEM) was from Gibco (Life Technologies, Carlsbad, CA). Primary antibodies used include anti-cleaved caspase-8, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP, anti-β-actin (Cell Signaling Technologies, Danvers, MA), anti-Fas Ligand, anti-alpha smooth muscle Actin antibody (Abcam, Cambridge, MA), anti-CD68 (AbD Serotec, Raleigh, NC), Rat Anti-Mouse Fas Ligand Monoclonal Antibody for neutralization, Rat IgG1 Isotype Control (R&D Systems, Minneapolis, MN), Anti-Mouse Fas Ligand FITC, Armenian Hamster IgG Isotype Control FITC (eBioscience, San Diego, CA). Fluorophore-conjugated secondary antibodies and 4′6-diamidino-2-phenyl-indole, dihydrochloride (DAPI) were purchased from Molecular Probes (Life Technologies, Carlsbad, CA). Horseradish Peroxidase (HRP)-conjugated Antibodies were purchased from Bio-Rad (Hercules, CA). In Situ Cell Death Detection Kit was from Roche Applied Science (Indianapolis, IN). siRNAs used in this study were Silencer Select siRNAs from Ambion (Life Technologies, Carlsbad, CA). Other chemicals and reagents if not specified were purchased from Sigma-Aldrich (St. Louis, MO).