Carboxyfluorescein succinimidyl ester (cfse)

THP-1 monocytic cells were purchased from ATCC. C666-1, an EBV positive undifferentiated nasopharyngeal cancer cell line was obtained from Professor Chan Soh Ha (NUS Immunology Centre). All cell lines were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS, 100 mg/ml penicillin/streptomycin (complete medium).
Human PBMCs were isolated from normal blood donor buffy coats using Ficoll density gradient (GE Healthcare). Monocytes (~98% purity) were isolated using anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).
To isolate THP-1 cells from C666-1 cells in our contact- co-cultures, THP-1 cells were labeled with 2.5 μmol/L CFSE (Molecular Probes, Eugene, OR) for 10 minutes at 25°C in a 0.1% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) buffer before co-culture. The CFSE+ (THP-1) and CFSE- (C666-1) fractions were isolated and analysed by flow cytometric sorting (Beckman Coulter).
To generate THP-1 macrophages, THP-1 cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma) for 48 hours. The cells were then washed 3 times with serum-free media to remove all traces of PMA and cultured in complete RPMI for another 48 hours before use in subsequent experiments.

Naïve splenocytes were labeled with 2.5 μM CFSE (Invitrogen Molecular Probes) and cultured in RPMI-1640 supplemented with 10% FBS, 20 U/mL recombinant IL-2 (Thermo Fisher Scientific), 1 μg/mL anti-CD3 (Thermo Fisher Scientific), and 5 μg/mL anti-CD28 (Thermo Fisher Scientific). Splenocytes were cultured alone or with freshly sorted PMN-MDSCs at the indicated ratio for 84 h at 37°C in a 5% CO₂ humidified atmosphere. Subsequently, CFSE dilution was assessed using a Cytoflex S flow cytometer (Beckman Coulter); splenocyte number was determined using the FlowJo Software (version 13; Tree Star).

Sorted CD4⁺CD8⁻CD25⁺ T cells (5 × 10⁴) from naïve and tolerant B6 mice bearing cardiac grafts were cultured for 14 days at 37°C in 5% CO₂ with 2 × 10⁵ BALB/c BMDCs in the presence or absence of interleukin (IL)-2 (1000 U/ml) and rapamycin (100 nM), alone or in combination. In some experiments, T cells were prelabeled with a solution of 5 mM carboxyflurescein diacetate succinmidyl ester (CFSE; Invitrogen, Carlsbad, CA, USA), followed by culture for 7 days with various stimuli. CFSE dilution was analyzed on a Beckman Coulter Epics XL.