Ix83 fluorescence microscope

The uptake of fluorescent beads was optimized using RAW 264.7 macrophages and 1 μm particle size fluorescent beads (yellow-green fluorescence, Cat# L4655, Sigma-Aldrich, USA). Macrophages were labeled with PKH26 (red fluorescence, Cat# MINI26-1KT, Sigma-Aldrich, USA) according to the manufacturer’s instructions and were seeded in 4-well glass chamber slides for 24 h. Five microliters of the bead solution were opsonized by resuspending with 5 mL of 50% FBS/PBS (phosphate-buffered saline) solution and incubated at 37 °C for 30 min. After opsonization, beads were seeded at increasing cell to bead concentrations [No beads (control), 1:12, 1:24 1:72, 1:150] and incubated at 37 °C for 2 h. Following engulfment, cells were washed 3 times with PBS to remove unengulfed beads. For fluorescence microscopy, the cells were fixed with 4% paraformaldehyde, washed with PBS, and mounted for fluorescence microscopy (Olympus IX83 fluorescence microscope). After optimization, the same experiment was repeated with BMDMs with a 1:150 cell to bead ratio.

BMDMs were plated at 5×10⁴ cells/well in 8-well glass slides. The macrophages were labeled with cell-permeant dye Calcein, AM (Cat# C1430, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions for 2 h. Apoptosis in H9c2 cells was induced by hydrogen peroxide (H₂O₂) treatment (Sigma-Aldrich, USA) for 4 h. The apoptotic cells were labeled with PKH26 (red fluorescence, Catalog-MINI26-1KT, Sigma-Aldrich, USA) according to the manufacturer’s instructions. Apoptotic H9c2 cells were overlaid on BMDMs (1:1 ratio of macrophage: apoptotic cells) and incubated for 2 h. After 2 h, cells were washed 4 times with ice-cold PBS to remove the unengulfed apoptotic cells. The cells were fixed with 4% paraformaldehyde, counterstained with DAPI (Thermo Fisher Scientific, USA), and mounted for fluorescence microscopy (Olympus IX83 fluorescence microscope). Phagocytosis was determined by counting cells containing engulfed red fluorescent apoptotic cells. A minimum of 300 macrophages was counted per well in triplicate. Data were represented as percent phagocytosis, i.e., the total number of cells with ingested apoptotic cells divided by the total number of macrophages counted times 100. In a separate phagocytosis experiment, BMDMs were stained with Flash Phalloidin^™ Green 488 (Cat# 424201, BioLegend, USA) to observe macrophage cell morphology.