Ly6c pe

Lungs from infected mice were used to generate single-cell suspension using mouse lung dissociation kit and the gentleMACS as per manufactures’ instructions (Miltenyi Biotec). Fungal cells were separated from mammalian cells via a 70%/30% discontinuous Percoll gradient centrifugation. Immune cells were stained with the fixable viability dye eFluor 455UV (eBiosecience) for 30 min at 4°C, washed with 1x PBS then fixed with a 2% paraformaldehyde (PFA) solution for 10 min at room temperature. Cell surface staining with antibody cocktail of mAbs specific to CD45-BV650, MHC-II-PECSF594, CD11c-APC, CD11b-BUV395, Ly6C-PE, Ly6G-FITC (all from BD Biosciences) was performed in FACS buffer containing 2% fetal calf serum, 2 mM sodium azide and anti-CD16/32 for 30 min at 4°C, washed then acquired on the BD Fortessa cell analyser (BD Biosciences). FlowJo software v10 (Tree Star) was used for data analysis.

After removing red blood cells with Ack lysis buffer, cells were stained with the following fluorescence-conjugated antibodies for 30 min at 4 °C: CD45R-APC/Cy7 (BioLegend, clone, dilution 1:400, Cat 103116), CD11b-PE/Cy7 (BioLegend, clone M1/70, dilution 1:400, Cat 101216), F4/80-APC (eBioscience, clone BM8, dilution 1:400, Cat 17-4801-82), Ly6G-PerCP/Cy5.5 (BD, clone 1A8, dilution 1:400, Cat 560602), Ly6C-PE (BD, clone AL-21, dilution 1:200, Cat 560592), CD11c-PE/Cy7 (eBioscience, clone N418, dilution 1:400, Cat 25-0114-82), and Siglec F-BV421(BD, clone E50-2440, dilution 1:400, Cat 562681). Cells were washed three times and analyzed on FACS Fortessa flow cytometer (BD Biosciences). Further analysis was implemented using Flowjo software (Treestar). Cell populations were defined as follows: macrophages in peritoneal cavities and spleen: CD45⁺CD11b⁺F4/80⁺; macrophages in bronchio-alveolar lavage fluid (BALF): CD45⁺CD11c⁺SiglecF⁺; monocytes: CD45⁺CD11b⁺Ly6C⁺; neutrophils: CD45⁺CD11b⁺Ly6G⁺.

The number of MDSCs, NKT cells, and T cells subsets were detected using FCM. Tumor tissues were cut into small fragments, digested with 0.25% Trypsin for 30 min, and then filtered using 70 μm cell strainers. This single-cell suspension was then centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the cell concentration was adjusted to 5 ×10⁸/ml. The cell suspension was then washed using a mouse tumor-infiltrating lymphocyte separation medium (CW0049S; CWBIO, Jiangsu, China) and centrifuged at 1500 rpm for 15 min to obtain the lymphocytes. Lymphocytes with a concentration of 2 × 10⁶/ml were collected and washed with PBS, followed by staining with anti-CD11b FTIC (No. 557397; BD, USA), anti-LY6G (No. 553989; BD, USA) and LY6C PE (No. 553126; BD, USA) (Gr-1), anti-CD3e FTIC (No. 46003 280; eBioscience, USA), anti-CD49a PE (No. 130107632; BD, USA), anti-CD4 FTIC (No. 11004282; eBioscience, USA), anti-CD8a PE (No. 11008182; eBioscience, USA), and anti-CD3 PerCP-CY 5.5 (No. 46 003280; eBioscience, USA), along with the appropriate isotype controls. The cells were analyzed using a flow cytometry FACSCalibur (BD, USA).

From each treatment group, mice not subjected to functional and histological analysis were used for flow cytometric analysis of cardiac inflammatory cell invasion. Mice were sacrificed on days 1, 3, and 7 after MI. Each time point, 3 hearts were harvested per group. Sham animals were used as controls to determine base line characteristics. Total cardiac cell numbers were determined with a Sysmex cell counter (Sysmex America, Inc. Mundelein, Illinois, US). Single-cell suspensions were stained for flow cytometry with primary antibodies before analysis using a FACSCanto II (BD Biosciences, San Diego, CA, USA). The following antibodies were used: anti–CD90-APC, 53–2.1,–B220-APC, RA3-6B2,–CD49b-APC, DX5,–NK1.1-APC, PK136,–Ly-6G-APC, 1A8, CD11b-eFluor 450, M1/70,–CD11c-FITC, HL3,–I-A^b -FITC, AF6-120.1,–Ly-6C-PE, AL-21,–CD11c-PE, HL3 (All above antibodies are from BD Biosciences),–F4/80-FITC, C1:A3-1 (ABD Serotec, Kidlington, UK). Monocytes were identified as CD11b high (CD90/B220/CD49b/NK1.1/Ly-6G) low (F4/80/I-A^b /CD11c) low Ly-6C high/low as previously described [26 (link),28 (link)]. Macrophages were identified as CD11b high, F4/80 high. Dendritic cells were identified as CD11b, I-A^bandCD11c high. Neutrophils were identified as CD11b, Ly-6G high. The analysis of the acquired data was done with FlowJo software version 7.6.1 (Tree Star Inc. Ashland, OR, USA).

After intracardiac injection of PBS, breast cancer tissues, lung metastases or lungs were harvested, minced and digested at 37°C for 45 min with DMEM medium containing collagenase type 1A (1.5 mg/ml), hyaluronidase (1.5 mg/ml), and DNase (20 U/ml). The digestion mixtures were filtered through 70 μm cell strainers. Single-cell suspensions were incubated with rat anti-mouse CD16/CD32 mAb (BD Biosciences), and then stained, washed and re-suspended in cold buffer (1%BSA, 0.1% NaN3 in PBS). 7AAD reagent (eBioscience) was added to the stained tubes (5 μl/tube) just before running the flow analysis. Flow cytometry data were acquired on a Gallios flow cytometer (Beckman, USA), and data were analyzed with Kaluza software (version 1.3). The appropriate, fluorochrome-conjugated, isotype-matched, control IgGs were used in all experiments. The following monoclonal anti-mouse antibodies were used: CD45-PE-Cy7, CD45-PerCP, CD45-BV421, Gr1-PerCP-Cy5.5, Gr1-APC, Gr1-APC-Cy7, CD11b-APC-Cy7, CD11b-BV510, Ly-6G-FITC, Ly-6C-PE (BD Biosciences) and F4/80-PE, F4/80-FITC, F4/80-APC (eBioscience).

Approximately 24 h after the last injection, mice were euthanized. The peripheral blood was collected and made into single-cell suspension. Erythrocyte lysate was used to get rid of red blood cells. Then cells were centrifuged and resuspended with 1 × phosphate-buffered saline (PBS). APC-CD11b (Biolegend, 101212), Gr-1-PerCP-Cy5.5 (BD, 552093), and Ly6C-PE (BD, 560592) fluorescent antibodies were used to incubate and stain cells for 45 min at 4°C. After washing, cells were resuspended with 300 μL PBS. The data were collected using a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo v7 software (Tree Star, Inc.).