Intracellular fixation permeabilization buffer

Heparinized peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood specimens by centrifugation over a discontinuous density gradient (Lympholyte-H; Cedarlane, Burlington, ON, Canada). Cell staining and flow cytometry were performed by using FACSCanto II (BD Biosciences, San Jose, CA, USA). All of the data were analyzed using FACSDiva software (BD Biosciences) and Flowjo Software (BD Biosciences) (18 (link)). We also examined expressions of intracellular IL-4 and IFN-γ using Cell Stimulation Cocktail pulse protein transport inhibitors^® and Intracellular Fixation & Permeabilization Buffer^® (Thermo Fisher Scientific, MA, USA) in a part of AA+AR patients. To detect secretory IL-10 of CD3^-CD19⁺CD24^hiCD27⁺ cells (Breg cells), we used IL-10 Secretion Assay Detection Kit (APC) ^® (Miltenyi Biotec B.V.&Co.KG, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) (19 (link), 20 (link)).

Before surgery, peripheral blood samples were collected from venous blood and preserved in heparin anticoagulant tubes at 4°C until experimentation (within 2 h). White blood cells were extracted by RBC Lysis Buffer (Thermo Fisher Scientific). RCC samples were obtained and examined just after surgical resection. At 37°C, freshly minced tumor samples were digested with collagenase IV (Sigma) and DNase I (Sigma), and then strained through a 70 μm strainer. The samples were then treated in RBC Lysis Buffer (Thermo Fisher Scientific). After blocking Fc receptors, single‐cell suspensions and white blood cells were stained separately for 30 min at 4°C with fluorescently labeled membrane marker antibodies. Intracellular proteins were stained with appropriate antibodies after being dissolved in Intracellular Fixation & Permeabilization Buffer (Thermo Fisher Scientific) according to the manufacturer's instructions. White blood cells and cell suspensions were stained with antibodies labeled with fluorochrome and maintained with cell staining buffer. Flowjo v10.0 was used to analyze BD LSRFortessaTM X‐20 (BD Biosciences) FACS data (Tree Star). Antibodies in detail are described in a previous publication.²⁶

Vaccinated mice were sacrificed, and the mesenteric lymph nodes (MLNs) and spleens were harvested. By pulverizing the tissue through a 40 μm cell strainer, lymph nodes and spleens were produced as single-cell suspensions. ACK buffer (HyClone) was used to lyse red blood cells, which were then centrifuged and suspended in RPMI 1640 medium (Basal Media) supplemented with 10% FBS (BI, Israel), and 1% streptomycin/penicillin. Single-cell suspensions were directly stained for flow cytometric analysis. Before intracellular cytokine staining, cells were stimulated in BFA and cell stimulation cocktail (Invitrogen) for 6 h. Next, the cells were stained for extracellular markers, fixed and permeabilized with Intracellular Fixation/Permeabilization Buffer (Invitrogen). Rat anti-mouse CD3 (clone 17A2), CD4 (clone GK1.5), IFN-γ (clone XMG1.2), IL-4 (clone 11B11), and IL-17 A (clone TC11-18H10.1) antibodies were purchased from BioLegend (San Diego, CA, USA). Flow cytometry was performed on a FACS Celesta flow cytometer (BD Biosciences, USA).

RAW264.7 cells were stained with anti-F4/80, iNOS and CD206 antibodies (Invitrogen). Briefly, cells were stained with cell surface marker prior to fixation and permeabilization with eBioscience Intracellular Fixation & Permeabilization Buffer (88–8824-00, Invitrogen). Cells were then stained with intracellular marker and analyzed using BD flow cytometer (BD Biosciences, San Jose, CA, USA).