Cd279 pd 1 fitc

One million cells of each tumor were transferred to a 96-well round-bottom, micro test plate and pelletized at 1500 rpm for 5 min (Beckman-Coulture Allegra X-14 Centrifuge). A fixable viability dye (eBioscience, eFluor 780) was used to identify live cells. The following antibodies were used for surface staining: CD3 APC (Biolegend, Clone: 17A2), CD4 BV510 (BD Bioscience Clone RM4–5), CD8a eFluor 450 (eBioscience, Clone: 53–6.7), CD279 (PD-1) FITC (eBioscience, Clone: J43), CD44 PECy5 (eBioscience, Clone: IM7), CD335 PECy7 (Biolegend, Clone: 29A1.4), CD11b AF488 (Biolegend M1/70), F4/80 BV421 (Biolegend BM8), CD206 PE (Biolegend C068C2), CD86 APC (Biolegend GL-1). Briefly, cells were stained with Fc blocking antibodies (TruStain FxX Biologend) for 10 min at 4 °C followed by cell surface antibodies in FACS Buffer (PBS with 2% FBS) for 30 min at 4 °C. Cells were pelletized at 1500 rpm for 5 min before re-suspending in 200 μL of FACS Buffer. Expression of T cell surface markers was measured by fluorescence cytometry (MACSQuant, Miltenyi Biotec) and analyzed by FlowJo software (Tree Star Inc.).

Zoledronic acid (ZA) was provided by Novartis Pharma AG Basel, Switzerland. Monoclonal antibodies against human CD25-PE, CD152 (CTLA-4)-PE, FOXP3-FITC, and CD279 (PD-1)-FITC were purchased from eBioscience, San Diego, CA, USA. CD194 (CCR4)-PerCP-CyTM 5.5, and CD69-FITC were purchased from BD Biosciences, San Jose, CA, USA. The MDA-MB-231 breast cancer cell line was purchased from the American Type Culture Collection (Food Industry Research and Development Institute, Taiwan) and was cultured with DMEM medium (Gibco®, Grand Island, NY, USA), containing 10% fetal bovine serum (FBS: Biological industries, Kibbutz Beit HaEmek, Israel). Conditioned media (CM) harvested from MDA-MB-231 cells consisted of DMEM supplemented with 2% FBS in which cells were cultured for 48 h. CM was collected, sterile filtered and stored in aliquots at −80 °C.