Pd l1 apc clone mih5

Single cell suspensions were prepared from freshly isolated CT26-HER2 tumors and spleens at sacrifice. The tumor specimens were minced and digested with collagenase (1 mg/mL) for 1 h at 37 °C. The resulting cell suspensions were passed through 70 μm cell strainer and rinsed with flow cytometry buffer (PBS+2% FBS). Spleens were processed directly by means of the cell strainer, and the red blood cells in splenic specimens were lysed by means of ACK buffer (150 mM NH₄Cl, 10 mM NaHCO₃, 1 mM EDTA), samples were pelleted and resuspended in flow cytometry buffer. For each sample, 2 × 10⁶ cells were blocked with α-CD16/32 Ab (clone 93, eBioscience-Thermo Fisher Scientific, Waltham, MA, USA), and then reacted with the antibodies CD4-FITC (clone GK1.5, eBioscience), CD8a-PE (clone 53–6.7, eBioscience), CD45-FITC (clone 30-F11, eBioscience), CD45-Percp-Cy7 (clone 30-F11, eBioscience), FoxP3-PE (clone 150d/e4, eBioscience), CD11b-FITC (clone M1/70, eBioscience), PD-L1-APC (clone MIH5, BD, Franklin Lakes, NJ, USA), and HER2-APC (clone 9G1D4B10, Sinobiological, Beijing, China). The data were acquired by means of BD C6 Accuri. Only the samples that provided at least 1 × 10⁵ events were included in subsequent analysis.

Single cell suspensions were prepared from freshly isolated HER2-LLC1 tumors and spleens at sacrifice. Tumors were minced in small pieces and digested with collagenase (1 mg/ml) for 1.5 h at 37°C. The resulting cell suspensions were passed through 70 μm cell strainer and rinsed with flow cytometry buffer. Spleens were processed as described above, and then treated as the tumor samples. Red blood cells in spleen and tumor specimens were lysed by means of ACK buffer, samples were pelleted and resuspended in flow cytometry buffer. Subsequently for each sample 2x10⁶ cells were blocked with α-CD16/32 Ab (clone 93, eBioscience), and then reacted with the antibodies CD4-FITC (clone GK1.5, eBioscience), CD8a-PE (clone 53–6.7, eBioscience), CD45-FITC (clone 30-F11, eBioscience), CD45-Percp-Cy7 (clone 30-F11, eBioscience), CD335-PE (clone 29A1.4, eBioscience), FoxP3-PE (clone 150d/e4, eBioscience), CD11b-FITC (clone M1/70, eBioscience), PD-L1-APC (clone MIH5, BD), CD141-PE (clone LS17-9, eBioscience) and CD69-PercP (clone H1-2F3, eBioscience). Data were acquired on BD C6 Accuri. Only samples which provided at least 100000 events were included in subsequent analysis.