Axiocam 702 monochrome camera

10⁷ Thymocytes were cultured in cDMEM in the presence of 1 μM of dexamethasone (Sigma) in a 3.5-cm culture dish at 37℃ in 5% CO₂ incubator for 8 hr. Apoptosis levels were assessed by PI (Biolegend) and Annexin V-FITC (Biolegend) staining. Typically, more than 80% of cells were Annexin V⁺. The dexamethasone-treated thymocytes were stained with 1 µg/mL pHrodo Red, SE (ThermoFisher) in PBS for 30 min at room temperature. The cells were washed two times with cDMEM and resuspended at 2×10⁶ cells/mL. 4×10⁴ sorted peritoneal and thymic macrophages were stained with 5-µM eFluor 450 (Thermo Fisher) in PBS for 10 min at 37℃, washed two times with cDMEM, and cultured in 96-well flat-bottom culture plate (Nunc) in 100 μL cDMEM at 37℃ in 5% CO₂ incubator. After 3 hr of attachment, the non-adherent cells were removed, and 200 µL (4×10⁵) apoptotic thymocytes were added to the macrophages. The cells were incubated at 37℃ in 5% CO₂ incubator for 2 hr. Fluorescent images were captured with AxioObserver 7 (Carl Zeiss) wide-field microscope equipped with Plan Apochromat 40 × NA = 1.0 objective (Zeiss) and AxioCam 702 monochrome camera (Zeiss) controlled by Zen 2.3 Blue (Zeiss) software. Image analysis was performed with Imaris 8.0.2 (Bitplane). Phagocytosis was scored by investigators blinded to the samples’ identities.

Ear and peritoneal cavity single-cell suspensions were enriched with CD117 conjugated magnetic beads (Miltenyi) and mounted with a cytospin (Cytospin 3, Shandon) onto Superfrost Plus slides (Thermo Scientific) under the following conditions: 500 rpm for 5 min. The slides were air-dried and stained with Giemsa (Merck) or Toluidine Blue O (Sigma) following standard procedures. Images were obtained with the Axio Observer 7 microscope equipped with Axiocam 702 monochrome camera (Zeiss).