Zen 2.3 blue

The confocal microscope images were obtained with a Zeiss Axio Observer inverted microscope (10x, 40x (oil) or 63x (oil) -objectives) equipped with LSM800 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany). TAMRA-FP, DAPI, and secondary antibodies were imaged with 561 nm (λ_ex 543 nm/λ_em 567 nm), 405 nm (λ_ex 353 nm/λ_em 465 nm), and 640 nm (λ_ex 653 nm/λ_em 668 nm) lasers, respectively. Secondary antibody used with bHABR (Figure S13) was imaged with 488 nm (λ_ex 495 nm/λ_em 519 nm) laser. ZEN 2.3 (Blue) and ZEN 2.3 lite softwares (Carl Zeiss Microimaging GmbH) were utilized for image processing and image analysis.

The immunostained brain was imaged dorso-frontally by using a confocal laser scanning microscope (LSM 800 Zeiss, Jena, Germany) equipped with a Plan-Neofluar 20×/0.5 objective. The sample was excited with a HeNe laser at 653 nm and the fluorescent emission passed through a 650 nm long-pass filter at a voxel size of 0.62 × 0.62 × 2 μm. The confocal images shown in this study were edited in ZEN 2.3 (blue edition, Carl Zeiss Microscopy GmbH, Jana, Germany).

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.