Annexin 5 staining

For cell surface staining, cells were incubated in the antibody cocktail for 20 minutes at 4°C in the dark. Samples were blocked using 30 μL normal rat serum (StemCell Technologies). Intracellular cytokine staining was performed using Cytofix/Cytoperm (BD) and intranuclear stain was performed using the FOXP3 staining buffer set (eBioscience, San Diego, California), both according to manufacturer’s instructions. Filipin-III was stained after overnight fixation of cells. Annexin-V staining was performed following the manufacturers’ instructions (ThermoFisher Scientific) by staining for 15 minutes at room temperature using Ca²⁺-containing Annexin-V buffer (diluted from 5X stock to 1X with ultrapure water). Cells were washed in room temperature also using Annexin V buffer prior to analysis. Flow cytometric analysis was performed using a 4-Laser Fortessa or 5-laser LSRII (BD) with FACSDiva software (BD). Fluorescence-activated cell sorting (FACS) was performed on a FACS Aria III (BD). Analyses were performed using FlowJo (BD Biosciences). For all flow experiments, a live/dead stain (ThermoFisher) was used to only assess live cells. Antibodies used for flow cytometry or cell sorting are listed in Table S2.

The healthy or dying cells were incubated with FITC-PspA, PspA fragments or FITC-Spn for 30 min. Dying cells were stained using annexin-V staining according to manufacturer’s instructions (Thermo Fisher). Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. Surface exposed GAPDH was stained using mouse anti-GAPDH antibody (Abcam) and then washed three times with PBS between the primary- and secondary-antibody incubation. As a negative control, FITC-labeled human serum albumin (Abcam, #8030) was used to evaluate non-specific binding of a host protein to healthy and dying cells in vitro and in the lungs. Cell nucleus was stained using 4,6-diamidino-2-phenylindole (DAPI; Thermo Fisher). Images were acquired using a Leica LMD6.

CD3+ cells were isolated from mouse spleens using EasySep kit from STEMCELL Technology. To evaluate the effects of EVs from L.monocytogenes-infected cells on apoptosis in bystander cells, resting, apoptosis-induced (low concentration of FasL, 5 ng/ml, Sigma-Aldrich, catalog no. F0427), or activated (Human T-Activator CD3/CD28 Dynabeads™, ThermoFisher Scientific catalog no 11161D) T lymphocytes were treated with supernatants from macrophages infected for 18h with L.monocytogenes (+/- GW4869). Apoptosis was evaluated by flow cytometry analysis Annexin V staining (ThermoFisher).
Murine splenic cells were stained with CD3-PE and T cells were sorted using FACS ARIA III sorter. Isolated murine T cells were activated with Mouse T-activator CD3/CD28 (11456D, ThermoFisher) for 48h and lysed using RIPA buffer (89901, ThermoFisher). The lysates were subjected to Western blotting.