Fix perm buffer

Splenocytes were prepared from mice as described above. 1 × 10⁶ splenocytes were resuspended in 100 μL staining buffer (BioLegend, USA) and incubated with anti-CD32/16 mAbs (BioLegend) for 10 min. Cells were washed and incubated in 100 μL staining buffer with surface antibodies for 30 min on ice in dark, then washed with staining buffer. Cells were fixed in formalin for 10 min at room temperature and washed with staining buffer. Finally, cells were resuspended in 500 μL staining buffer for flow cytometry.
Intracellular cytokine labeling was performed after surface molecule staining as described above. Splenocytes were seeded in 96-well plates, stimulated with BCG protein (25 μg/mL) and then incubated at 37°C for 12 h. Brefeldin A (BioLegend, USA) was added into wells for another 12 h of incubation at the final concentration of 5 μg/mL. Cells were then washed and labeled with cell surface antibodies as described above. Then fixed cells were permeated with 1 mL Fix/Perm buffer (BD, USA) for 20 min. Cells were washed with 1 mL 1 × Perm/Wash buffer (BD, USA) twice, then pelleted and resuspended in 100 μL 1 × Perm/Wash buffer. Cells were incubated with titrated intracellular antibodies for 30 min at room temperature. After washing thrice, cells were resuspended in volumes of 500 μL staining buffer for flow cytometry.

We prepared and stained single-cell suspensions from both kidneys as described previously [11 (link), 15 (link), 39 (link)]. The antibodies used for FACS were as follow: fluorescein isothiocyanate-conjugated rat anti-mouse CD11b (#557396, Becton Dickinson (BD), San Jose, CA, USA), Alexa Fluor 647-conjugated rat anti-mouse F4/80 (#565853, BD), phycoerythrin-conjugated rat anti-mouse TNF-α (#554419, BD), and phycoerythrin-conjugated rat anti-mouse IL-10 (#554467, BD). To examine intracellular cytokine expression, kidneys were incubated in RPMI 1640 containing GolgiStop™ solution (#554715, BD) for 8 h in 5% CO₂ at 37°C. The cells were washed in phosphate-buffered saline, suspended in FACS buffer (#554656, BD) with protein-block (Fcg III/II Receptor, 1:100 dilution, BD, #553141), and incubated with cell surface markers for 30 min on ice. Then, the cells were fixed and permeabilized using Fix/Perm buffer (#554715, BD). After the staining of intracellular cytokines, the cells were washed twice in Fix/Perm buffer, and suspended in FACS buffer to allow the resealing of the permeabilized membranes. We collected 1.0 × 10⁵ to 5.0 × 10⁵ total kidney cells using FACSCalibur (BD), and analyzed the data using FlowJo software 9.3 (Tree Star, Palo Alto, CA, USA).

All flow cytometry was performed using Becton-Dickinson Canto or LSR-II flow cytometers. For innate cell surface phenotyping, PL or MLN were stained with a combination of antibodies to Siglec-F (E50-2440), CD11b (M1/70), F4/80 (BM8), Ly6G (1A8) and Ly6C (AL-21 or HK1.4). Following fixation and permeabilisation with the Foxp3 staining kit (eBioscience) cells underwent intracellular staining with RELM-α (RnD Systems) followed by zenon anti-rabbit A647 (Invitrogen) and FITC-conjugated anti-human Ki67 (BD Biosciences). For intracellular cytokine staining of monocytes, 0.5−1 × 10⁶ PL cells were incubated with 10 μg/ml Brefeldin A for 4 hr. Following cell surface staining as above, and fixation and permeabilization with Fix/Perm buffer (BD Pharmingen), cells underwent intracellular staining with anti-IL-13 (JES10-5A2). For intracellular staining of lymphocytes, MLNCs were incubated with 0.5 μg/ml PMA and 1 μg/ml ionomycin for 1 hr before the addition of 10 μg/ml Brefeldin A for a further 3 hr. Staining was performed by re-suspending cells in a combination of Abs to CD4 (GK1.5), ICOS (DX29), and the following combination to define Lin^–: CD3 (17A2), CD5 (53–7.3), CD8α (RPA-T8), CD49b (DX5), CD11c (HL3), F4/80 (BM8), CD19 (eBio1D3), Gr-1 (RB6-8C5), TCRβ (H57-597) and CD11b (M1/70).

For cardiac differentiation assessment, RFP6-, iSMAD1- and iSMAD2-EBs were harvested at Day 7+3. For the RFP6-ESC clonal cell line, flow cytometry analysis was performed on single cell suspension of live cells, and cardiomyogenesis quantified by the amount of RFP protein. For the inducible clonal cell lines iSMAD1-ESCs and iSMAD2-ESCs, following trypsinization, single-cells were fixed with Fix/Perm buffer (BD Biosciences) and stained with the primary mouse antibody to TNNT2 (NeoMarkers MS295R7), followed by incubation with a secondary AlexaFluor 647 conjugated chicken anti-mouse IgG (Life Technologies). A mouse-IgG1 antibody was used as the isotype control (Life Technologies MG100). All data were acquired using a FACSCanto (BD Biosciences), counting a minimum of 5000 to 10,000 events.