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The tumor tissues were digested for one hour at 37 °C with collagenase and hyaluronidase (1 mg/mL) to prepare the single-cell suspension. The lymph nodes and spleen were added with PBS (1 mg/mL) and ground to prepare a single-cell suspension. The cells were incubated with PBS with 0.2% BSA on ice for 30 min, then washed with PBS. The cells were stained using CD8-PE, IFN-γ-PE-Cy7, CD11C-FITC, CD86-APC, CD206-PE, F4/80-FITC, CD44-FITC, or CD62L-APC (BD Biosciences, Franklin Lakes, USA) for flow cytometry assay according to a standard procedure.

Mice infected or uninfected with MHV-3 were sacrificed at 3 and 5 dpi. The spleens were removed and processed as previously described [25 (link)]. The lungs were removed and processed as previously described [26 (link)]. Purified cells from the lungs and spleen were plated and incubated with brefeldin A (10 μg/mL) (Invitrogen, Waltham, MA, USA) for 3 h at 37 °C in the presence of 5% CO₂. Cells were blocked with Fc Block (antibody CD16/CD32 in PBS/BSA 1%), followed by stain using specific combinations of antibodies for cell surface molecule labeling: CD3, CD11b (APC-Cy7); CD4, Ly6C (PE-Cy7); CD8, Ly6G, SinglecF (BV421); CD25, CD45 (PerCP-Cy5.5); F4/80 (FITC); CD11c (V500), and isotype controls (all from BD Biosciences). For intracellular staining, the following antibodies were added: IFNγ (Alexa 488), IL-17, forkhead box P3 (FOXP3) (PE), TNF (PE), and IL-10 (APC). A total of 30,000 cells (events) were acquired using a FACSCanto II cytometer (Becton, Franklin Lakes, San Jose, CA, USA) and analyzed using the FlowJo software (version 10).

Peripheral blood, spleens, and mesenteric lymph nodes (mLN) were collected 24 h after CLP surgery or sham surgery to obtain single cells for flow cytometry. Cells were stained for 30 min at 4°C with the following antibodies: CD45-Percp, CD3-FITC, CD4-Percp, CD8-APC, B220-PE, CD11c-APC, CD11b-FITC, F4/80-PE, and Ly6G-PE. Following incubation, red blood cells were lysed and washed 3 times with a FACS buffer before collecting data. Immunophenotyping analysis of the BMDMs was conducted using a flow cytometry technique. Briefly, 100 μl of cell suspension was incubated for 5 min with 5 μl of Fc blocker (Innovex, USA). Then, 1 μl of monoclonal antibodies (CD11b-FITC, F4/80-PE, F4/80-FITC, MHCII-FITC, CD80-PE, CD80-APC, CD16/32-Percp-cy5.5, and CD64-APC, BD, USA) were added and incubated at 4°C for 30 min and washed 3 times with the FACS buffer. All samples were analyzed using a FACSCalibur flow cytometer (BD, USA).

Mice infected with PbA were euthanized at 5 dpi. The brains and spleens were removed and processed according to the methodology described by Brant et al. [37 (link)]. Lungs were removed and processed according to the methodology described by Claser et al. [12 (link)]. Purified cells from the brain, lung, and spleen were plated and incubated with brefeldin A (10 μg/mL) (Invitrogen, Waltham, MA, USA) for 3 h at 37 °C in the presence of 5% CO₂. The cells were blocked with Fc Block (antibody CD16/CD32 in PBS/BSA 1%), followed by staining with specific combinations of antibodies for cell surface molecule labeling: CD3 and CD11b (APC-Cy7); CD4 and Ly6C (PE-Cy7); CD8, Ly6G, and SinglecF (BV421); CD25 and CD45 (PerCP-Cy5.5); F4/80 (FITC); CD11c (V500); and isotype controls (all antibodies from BD Biosciences, Franklin Lakes, NJ, USA). For intracellular staining, the following antibodies were added: IFN-γ (Alexa 488), IL-17A, Foxp3 (PE), TNF (PE), and IL-10 (APC). A total of 30,000 cells (events) were acquired using a FACS Canto II cytometer (BD Biosciences) and analyzed using FlowJo software (ver. 10). Our gating strategy is shown schematically in Supplementary Figure S1 (for innate immune cells subset composition) and Supplementary Figure S2 (for adaptative immune cells subset composition).

Analysis of immunological spleen-cells of mice treated or not with STM was conducted by flow cytometry 30 days PTI. The erythrocytes were lysed using lysis buffer before staining. Cells were immunostained with the following antibodies: FITC-CD3, PE-Cy7-CD8, PE-CD4, PE-Cy7-CD220, FITC-F4/80, FITC-ly6G, PE-Cy7-Ly6C, Pe-γδ (BD Pharmingen, San Diego, USA). Cells were harvested, washed with PBS-5% FBS and stained with antibodies for 15 min in the dark at 4°C. Cells were fixed for 20 min with 1% paraformaldehyde/PBS. A gate was set within the lymphocyte-monocyte populations in the FSC–SSC dot-plot and 50, 000 events were collected.