Carboxyfluorescein succinimidyl ester (cfse)

MSCs were stimulated with TNF-α (10 ng/ml) for 24 h to prepare T-MSCs. OTX008 (60 μM) was added into MSCs 1 h before TNF-α addition to inhibit Galectin-1 expression. MSCs or carboxyfluorescein succinimidyl ester (CFSE)-marked MSCs were added into the mouse peritoneal macrophage culture environment (1:10) directly or with transwell for 24 h. MSCs were sustained with CFSE (2.5 μg/1 × 10⁷ cells) purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 10 min to obtain CFSE-marked MSCs. MSC-derived exosomes (25 μg/ml) were added into the macrophage culture environment for 24 h. Recombinant human Galectin-1 (1 μg/ml) was used to treat mouse peritoneal macrophages for 24 h. Ruxolitinib (1 μmol/L), BAY 11-7082 (1 μmol/L), VX-11e (100 nmol/L), and rapamycin (10 μmol/L) were used to pretreat mouse peritoneal macrophages for 1 h.

To measure the immunosuppressive capacity of human BMMSC, the isolated human PBMCs were incubated with 5 µm carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, #C34570) for 10 min at 37 °C. FBS was then added, and the cells were incubated for 5 min. The cells were washed twice with PBS and counted. The CFSE‐labeled PBMCs were cocultured with BMMSC at a cell ratio of 1 BMMSC per 10 PBMCs in 1640 medium containing 10% FBS, 1 µg mL⁻¹ anti‐human CD3/CD28 antibodies (PeproTech, #05121‐25, #10311‐25) and 100 IU mL⁻¹ IL‐2 (Prosperich, #CSBSJ‐IL‐2). For the measurement of mouse BMMSC, the CFSE‐labeled splenocytes were cocultured with mouse BMMSC at a cell ratio of 1 BMMSC per ten splenocytes in 1640 medium containing 10% FBS, 1 µg mL⁻¹ anti‐mouse CD3/CD28 antibodies (PeproTech, #05112‐25, #10312‐25) and 10 ng mL⁻¹ IL‐2 (PeproTech, #212‐12). On the fourth day, the cells were collected, stained with live/dead dye 780, and detected by flow cytometry.

Teff cells were stained with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) and suspended in complete RPMI-1640 medium. CFSE-labelled Teff cells (5 × 10⁴ cells/well) were stimulated with Dynabeads^® Mouse T-Activator CD3/CD28 (Life Technologies, 2 μl/well) for 72 h in the presence of Treg cells at various ratios on 96-well round-bottom plates (BD Biosciences). For inhibition of TGF-β1, CFSE-labelled Teff cells were stimulated with Dynabeads^® Mouse T-Activator CD3/CD28 for 72 h with or without recombinant human TGF-β1 (PeproTech. INC., Rocky Hill, NJ, USA). Then, CFSE fluorescence intensity was measured by FACS LSRII to determine the proliferation of Teff cells.

HSR-GBM1 and 040821 cells were transduced with lentiviruses encoding control pLenti6 vectors or JAG1 constructs. At least 5 × 10⁵ cells in each line (premix cells) were collected per line for RNA extraction. 5 × 10⁶ control vector cells were stained with the viable dye Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) according to manufacturer's instructions (Invitrogen, Carlsbad, CA) and co-cultured with the same number of JAG1 overexpressing cells in a T75 flask for 17 h. Co-cultured cells were then sorted into CFSE-positive (pLenti6) or CFSE-negative (plenti6-JAG1) populations by flow cytometry. Similarly, 6 × 10⁶ control vector and 6 × 10⁶ JAG1 XPA3 cells were co-cultured in a T75 flask for 17 h prior to sorting. The experiment was performed at least twice in each cell line.

Canine PBMCs were thawed and rested for 1 h at 37°C prior to CFSE labeling by incubating with 10mM CFSE (Thermo Fisher) for 10 min at 37°C while protected from light. Cells were washed twice in RPMI1640 media and counted. 2x10⁵ CFSE-labeled PBMCs were plated per well in 200 μL of complete culture media: RPMI 1640 (Invitrogen) supplemented with 10% FBS (Fisher biotech), 20mM HEPES (Invitrogen), 0.05 mM 2-mercaptoethanol (Thermo Fisher), and 100 U/ml penicillin (Invitrogen) with or without KLH protein (20 μg/mL, Imject mcKLH Subunits, Thermo Fisher). 72 h later, cells were analyzed by flow cytometry for CFSE dilution and intracellular cytokine staining using a panel of canine antibodies derived from OMIP-065.⁶⁶ (link) Flow cytometry antibodies are detailed in the key resources table.

The in vitro suppressive capacity of Breg^IL-33 on B responder cell proliferation and division was determined by the ³H-thymidine uptake and CFSE dilution assays. Respectively, fixed numbers (10⁵) of B responder cells (CD19⁺CD23⁺) were cultured, in the presence or absence of anti-CD40 (2.5 μg/ml, Enzo Life Sciences, UK), anti-IgM (5 μg/ml, Thermo Scientific, UK) or LPS (0.5 μg/ml, Sigma–Aldrich, UK), with titrated doses of Breg^IL-33 (CD19⁺CD23⁻) isolated from IL-33-treated WT or IL-10^−/− mice as described above. In some experiments, purified Breg^IL-33 were first primed by the anti-CD40 antibody (2.5 μg/ml, 5 h) before adding them to the responder cells. For the cell proliferation assay, 1 μCi/well ^[3H]thymidine (Amersham, UK) was added for the last 16 h, and the thymidine incorporation determined using a scintillation counter (PerkinElmer, MA, USA). For the CFSE dilution assay, responder cells were pre-labelled with CFSE (Molecular Probes, NY, USA), before co-culturing and stimulation, and cell division (CFSE dilution) measured by flow cytometry.

Fresh splenic conventional T cells were sorted as CD4⁺CD25⁻ from C57BL/6 mice. Tregs were sorted from Foxp3^eGFP as CD4⁺CD90.1⁺GFP⁺. For carboxyfluorescein succinimidyl ester (CFSE) labelling, purified CD4⁺ T cells were resuspended in 10μg/ml of CFSE (Molecular Probes) for 10⁷ cells for 10min at 37°C in dark in RPMI-1640 0% FBS and were washed in cold RPMI-FCS 10% to neutralize CFSE action. 4×10⁴ CFSE-labeled CD4⁺CD25⁻ GFP^- T cells were incubated with 10⁵ irradiated splenocytes (2000 rad) with or without addition of Treg cells at the indicated ratios, and stimulated with 0.5μg/ml soluble anti-CD3 (2C11) for 72 h. Dividing cells were identified by CFSE dilution on FACS analysis.