Annexin 5 staining kit

Apoptotic cells were analyzed by an annexin V staining kit (Thermo Fisher Scientific). Briefly, the cells were harvested and washed twice with cold PBS. The cells were resuspended in 1× binding buffer and stained with annexin V-FITC reagent for 15 min at room temperature. Then, the cells were washed with 1× binding buffer and incubated with propidium iodide (PI) for 5 min on ice. The stained cells were analysed by flow cytometry with a BD FACSCalibur cytometer (BD Biosciences, San Diego, CA, USA).

BMDMs were pretreated with Tm (1 mg/ml) for 6 h prior to stimulation with LPS (100 ng/ml) 10 h. Cell apoptosis was measured by an Annexin V staining kit according to the manufacturer's instructions (eBioscience). Briefly, BMDMs in non-TC (tissue culture) treated dishes were detached by trypsinization and washed with PBS. Cells were resuspended in binding buffer and stained with Annexin-V-FITC solution. The proportion of Annexin V+ cells was calculated in the gated single cell population by BD LSRortessa flow cytometer with FlowJo software. The average fluorescence intensity of the Annexin V+ cells in each group was obtained in the gated Annexin V+ cell population and analyzed by FlowJo software. For PI immunofluorescence microscopy assay, the cells were washed with cold PBS and incubated with Hochest 33342 (10 μg/ml) and PI (1 mg/ml) solution (Solarbio) for 15 min. The cells were observed under Zeiss Vert.A1 microscope. Fluorescence images were captured with Zeiss Axiocam 503 color CCD camera controlled with ZEN software (ZEISS).

All the antibody information is provided in supplementary table 1. Annexin V were detected by Annexin V staining kit from ebioscience (catalog# 88-8103-74) according to manufacturer’s protocol. Flow cytometry analyses were performed with CyAn Immunocytometry system (DAKO Cytomation, Fort Collins, CO), Attune NxT Flow Cytometer (ThermoFisher Scientific) and BD LSRFortessa (Franklin Lakes, NJ), the resulting data were analyzed with FlowJo software (Tree Star, Ashland, OR). Cell sorting was performed with a BD FACS Aria SORP sorter at the City of Hope FACS facility. The sorted cells were used for transfer experiments.