Goat f ab 2 anti mouse igg h l

Bone marrow-derived macrophages (BMDM) were isolated and cultured as described [24 ]. To stimulate BMDM, 10⁶ cells were incubated for 30 min at room temperature with 10 μg/mL F(ab′)₂ fragments of a MIP8a antibody, generated through digestion with pepsin (Sigma-Aldrich). After washing twice with RPMI 1640, goat F(ab’)₂ anti-mouse IgG (H+L) (SouthernBiotech, Birmingham, AL) was added for 2 h at 37°C to crosslink CD89. As positive controls 2 U/mL IFNγ (PeproTech, Rocky Hill, NJ) and 10 ng/mL LPS (Sigma-Aldrich, St Louis, MO) added to 10⁶ BMDM cells for 4 h at 37°C were used. The negative control lacked the cross-linking Ab. Subsequently, CD89-specific intracellular ROS production was measured by flow cytometry using a cell-based ROS assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s instructions. Macrophages were trypsinized, washed with PBS, and then incubated with DCFH-DA at a final concentration of 10 μM for 30 min at 37°C. Fluorescence was analyzed on a FACSVerse (BD Pharmingen). Intracellular ROS levels were expressed as the average DCF fluorescence intensity of the cells.

RPMI medium 1640 (1x) + GlutaMAX was obtained from Gibco (Live Technologies) and Fetal Bovine Serum (FBS) from Biochrom AG (Berlin, Germany), and propidium iodide (PI), 4′-6-diamidino-2-phenylindole (DAPI), bicinchoninic acid solution, and gentamicin were obtained from Sigma-Aldrich Quimica, S.A. (Madrid, Spain). Metformin and the annexin V-FITC Apoptosis Detection Kit were purchased from Calbiochem (Darmstadt, Germany) and the XTT Cell Proliferation Kit II and RNase from Roche Molecular Biochemicals (Indianapolis, IN, USA). The Vybrant FAM caspase-3 and caspase-7 assay kit and DiOC₆(3) were obtained from Molecular Probes (Live Technologies).
The rabbit polyclonal anti-cyclin A (1 : 500, sc-751), anti-cyclin E (1 : 500, sc-481), anti-PKCε (1 : 200, sc-214), and anti-PKCδ (1 : 200, sc-213) antibodies and the mouse monoclonal anti-cyclin B1 (1 : 500, sc-245) and anti-cdc2 p34 (1 : 500, sc-54) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit polyclonal anti-LC3B (1 : 3000, ab51520) and the HRP-conjugated goat secondary polyclonal antibody against rabbit IgG-H&L (1 : 3000, ab6721) were purchased from Abcam (Cambridge, UK). Goat F(ab′)₂ anti-mouse IgG (H+L) was obtained from Southern Biotech (1032-05) and the rabbit anti-actin (1 : 200, A2066) antibody was from Sigma-Aldrich Quimica, S.A. (Madrid, Spain).