Facs canton 2

Mononuclear cells from pleural effusion or peripheral blood were isolated by Ficoll-Hypaque (Huajing Biology Co., Shanghai) density gradient centrifugation. 1×10⁵ cells were stained with APC-Cy7 labeled anti-human CD14 (Biolegend) and PE labeled anti-human CD163 (Biolegend) antibodies. Dead cells were stained using 7-AAD (BD Biosciences). After incubation for 15 min on ice in the darkness, the cells were analyzed by FACSCanton II (BD).
To investigate the effect of PA-MSHA on CD163+ macrophages, the percentages of CD163+ macrophages in MPE before and after treatment of PA-MSHA in clinic and in vitro were analyzed by flow cytometry as above method, respectively.

Venous blood was collected aseptically from patients and healthy volunteers. Red blood cells in the human peripheral blood were lysed and washed twice with phosphate-buffered saline (PBS). In addition, single-cell suspensions of blood and liver of mice were obtained 12 h after ConA administration by using mechanical and enzymatic dispersion as described previously^{15 (link)}. In general, the peripheral blood and liver were harvested. Red blood cells in the peripheral blood were lysed. A single-cell suspension of the liver was mechanically disrupted and then enzymatically digested with 1 mg/mL collagenase IV. Then, 1 × 10⁶ freshly prepared cells were suspended in 100 μl of PBS and stained with different combinations of fluorochrome-coupled antibodies. Data were acquired by flow cytometry on a FACS Canton II (BD Biosciences) or NovoCyte (ACEA) and analyzed by FlowJo software.

Naïve Balb/c and ΔdblGata mice were euthanized and their peritoneal cavity was flushed by injecting 10mL of PBS in 10% FBS into peritoneal cavity, massaging the abdomen, and then drawing out the fluid. Cells were kept on ice, centrifuged at 500g for 5 mins, and supernatant removed. Pellets were lysed with 5mL Red Blood Cell Lysis Buffer (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes at room temperature, 5mL of RPMI 1640 media added to neutralize lysis, and centrifuged for 5 mins. Pellet was re-suspended in media and counted. Cells were stained with phycoerythrin (PE)- conjugated anti-c-Kit (BD Pharmingen, Mountain View, CA, USA), PE-cy7-conjugated anti-FcεRIα (Biolegend, San Diego, CA, USA) and biotinylated anti-T1/ST2. They were subsequently counterstained with Pacific Blue labelled streptavidin. Analysis was performed on FACS Canton II (BD Bioscience, Mountain View, CA, USA) and analyzed using Flowjo (Flowjo Software, Ahsland, OR, USA).

Lamina propria cells from SI of ΔdblGata or WT mice were stained with phycoerythrin (PE)- conjugated anti-c-Kit, fluorescein isothiocyanate (FITC)-conjugated anti-β7 integrin, Horizon V500-conjugated CD4, APC-Cy-7-conjugated anti-CD3e (BD Pharmingen, Mountain View, CA, USA), allophycocyanin (APC)-conjugated anti-IL-17RB, PE-Cy7-conjugated anti-FcεRIα (Biolegend, San Diego, CA, USA) and biotinylated anti-T1/ST2. Subsequently, cells were counterstained with PerCP-Cy5.5-conjugated monoclonal antibodies against lineage (Lin) markers (CD11b, CD11c, CD45R (BD Pharmingen, Mountain View, CA, USA) CD8α, Ly6G, and Pacific Blue labelled Streptavidin (Biolegend, San Diego, CA, USA)) before analysis with a FACS Canton II (BD Bioscience, Mountain View, CA, USA). CD4⁺ Th₂ cells were identified as Lin^-, CD3⁺, CD4⁺, and IL17RB⁺ populations. ILC2 cells were identified as Lin^-, CD3^-, CD4^-, 1L17RB⁺, and c-Kit^- populations. MMC9 cells were identified as Lin^-, CD3^-, CD4^-, 1L17RB^-, c-Kit⁺, and FcεRIα⁺ populations as previously described [38 (link)]. All cytometric data was acquired using BD FACSCanto II and data analysis was performed using Flowjo software (FlowJo, Ashland, OR, USA).

PBMCs were isolated from heparinized blood samples by Ficoll–Hypaque density-gradient centrifugation. Peripheral CD4+ T cells of controls were isolated from PBMCs by human CD4 microbeads (Miltenyi Biotec, Palo Alto, CA, USA) according to the manufacturer’s instructions. The purity of CD4+ T cells was detected using FACS Canton II and FACS Diva software (BD Company, Franklin Lakes, NJ, USA) after staining with anti-human CD4 antibodies (BD Company, USA). The result showed that the purity was >92% in each experiment. PBMCs were cultured for 72 h with or without rhIFN-α 2a (PBL Biomedical Lab, Piscataway, NJ, USA), anti-IL-10 (R&D Systems) at a concentration of 1 × 10⁶ cells/ml in combination with anti-CD3 (5 mg/ml) and anti-CD28 anti- bodies (1 mg/ml) (eBioscience, San Diego, CA, USA). CD4+ T cells (1 × 10⁶ cells/ml) were cultured for 72 h with or without rhIFN-α 2a (1 mg/ml) in combination with anti-CD3 (5 mg/ml) and anti-CD28 antibodies (1 mg/ml) (Miltenyi Biotec,Germany). Supernatants from these cell cultures were used for detecting the concentration of IL-17 and IL-10.

For surface marker staining (Ning et al., 2016 (link)), single cell suspensions from mice tumors were stained with fluorescein-conjugated mouse-specific antibodies against CD11c, CD11b, F4/80, Gr-1, PD-1, PD-L1, CD45, CD3, CD4 or CD8 (Biolegend) at 4°C for 30 min in the dark. After the cells were washed twice with PBS, they were either directly measured or, for intracellular staining, fixated and permeabilized with Foxp3 (Biolegend) to stain nuclear factors. Intranuclear antibodies were then incubated at room temperature for 30 min in the dark. This was followed by washing and flow cytometry, which was performed on a FACS Canton II (BD Biosciences). Results were analyzed using FlowJo version 10 (Tree Star).
Immune cell subsets were characterized using the following sets of markers: DCs (CD11c⁺/CD8⁺), macrophages (CD11b⁺/F4-80⁺), myeloid-derived suppressor cells (MDSCs: CD11b⁺/Gr-1⁺), CD8⁺ T cells (CD45⁺/CD3⁺/CD4^-/CD8⁺), CD4⁺ T cells (CD45⁺/CD3⁺/CD8^-/CD4⁺), and Tregs (CD45⁺/CD3⁺/CD4⁺/Foxp3⁺).