P35361

For in vitro experiments, briefly,⁴² pH-sensitive latex beads, pH-sensitive Escherichia coli (E. coli; P35361, Invitrogen), PKH26-labeled RBCs, or propidium iodide (PI)-labeled dying cancer cells were incubated with cells for 20 min, respectively. Phagocytes were washed and fixed with 4% paraformaldehyde (PFA; P1110, Solarbio), washed, and immunostained. Z-stack images were captured with a confocal microscope (TCS SP8 STED, Leica, Germany). The beads number was calculated by the number of engulfed beads per phagocyte, and the engulfment (%) was calculated by the percentage of engulfed phagocytes in total phagocytes in each field. For in vivo experiments, briefly,⁸ (link) PKH26-labeled RBCs (1 × 10⁶) in 2 μL phosphate buffered solution (PBS; P1010, Solarbio) were injected into the left mouse striatum at 0.4 μL/min (coordinates: x = 2.0 mm, y = 0.8 mm, z = −3.15 mm). Mice were sacrificed and brain sections were used for immunofluorescence staining. Four random perihematomal areas on 6 brain sections at 3 slides per animal were used and quantified by ImageJ software. The RBC number was calculated by the number of engulfed RBCs per microglia/macrophage.

For the phagocytosis assay in the acute brain slice culture, brain slices were prepared as previously described and incubated with pHrodo Red E. coli BioParticles Conjugate for Phagocytosis (0.25 mg/ml; P35361, Invitrogen) or pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (P35366, Invitrogen) in the Leibovitz’s L-15 medium (11415064, Gibco) containing 10% FBS.
For the phagocytosis assay in cell suspensions, microglia suspensions were prepared as previously described and incubated with pHrodo Red E. coli BioParticles Conjugate for Phagocytosis (0.8 mg/ml; P35361, Invitrogen) in the Leibovitz’s L-15 medium (11415064, Gibco) containing 10% FBS for 1 hour at 30°C. After incubation, cell suspensions were analyzed in a FACSAria III sorter (BD Biosciences).