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Flow cytometry

DS Dong Shao
YC Yaping Chen
HH Hao Huang
YL Yingting Liu
JC Junjun Chen
DZ Dawei Zhu
XZ Xiao Zheng
LC Lujun Chen
JJ Jingting Jiang
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Tumor tissues from the left tumors were collected and digested with Liberase TL (Catalog No. 05401020001, Roche) and DNase I (Catalog No. 10104159001, Roche) for 30 min at 37 °C, and prepared into single-cell suspension as reported in our previous study [12]. Antibodies used in staining and flow analysis included CD45 (Clone 30-F11), CD3 (Clone 17A2), CD4 (Clone GK1.5), CD8 (Clone 53–6.7), NK1.1 (Clone PK136), LAG3 (Clone C9B7W), Foxp3 (Clone 3G3), CD11b-BV650 (Clone M1/70), MHC-II (Clone AF6-120.1), CD11c (Clone N418), CD206 (Clone C068C2), F4/80 (Clone BM8) and CD103 (Clone M290). Intracellular cytokine staining was also performed as described in previous study [12]. Following stimulation, the cells were labeled with antibodies against surface markers, fixed, and permeabilized using the Invitrogen Fixing/Permeabilization Solution kit’s manufacturer’s instructions. Antibodies against TNF-α (Clone MP6-XT22) and IFN-γ (Clone XMG1.2) were used to stain the fixed cells. A BD FACS Aria II flow cytometer was used to collect data, which were then analyzed using FlowJo software.

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