Single cell suspensions of mouse tumors were prepared for flow cytometric analysis. Cells were blocked with anti-mouse CD16/32 (BioLegend) and surface stained with indicated markers. Live/dead cell discrimination was performed using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies). To evaluate change in the frequency of myeloid cells and MDSCs in the TME by ISIM, we used 12-color flow cytometry gating strategy (18 , 22 (link)). The following Ab were used; anti-CD45 (clone 30-F11, Invitrogen), anti-CCR2 (R&D systems), anti-Ly6C (clone HK1.4, BioLegend), anti-CD11b (clone M1/70, BD Biosciences), anti-I-Ab (clone AF6-120.1, BD Biosciences), anti-PD-L1 (clone MIH5, BD Biosciences), anti-CD62L (clone MEL-14, BioLegend), anti-CX3CR1 (clone SA011F11, BioLegend), anti-F4/80 (clone BM8, BioLegend), anti-CD24 (clone M1/69, BD Biosciences), and anti-Ly6G (clone 1A8, BioLegend). LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific)-stained cells were excluded from analysis. Samples acquired on LSRII or Fortessa (BD Biosciences) cytometers were analyzed with FlowJo software (Treestar).
Protocol detail
Flow Cytometric Analysis of Tumor Microenvironment
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Corresponding Organization :
Other organizations : Roswell Park Comprehensive Cancer Center, University at Buffalo, State University of New York
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Variable analysis
independent variables
- None explicitly mentioned
- Frequency of myeloid cells and MDSCs in the TME
- Cells were blocked with anti-mouse CD16/32 (BioLegend)
- Live/dead cell discrimination was performed using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies)
- LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Thermo Fisher Scientific)-stained cells were excluded from analysis
- Positive control: None mentioned
- Negative control: None mentioned
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