Cytomics fc500 mpl flow cytometry system

Human monoclonal antibodies against HLA-DR, CD4, CD33, CD11b, CD45, CD8, CD25, CD3, CD16, FOXP3, CD14, CD15, CD39, CD73, CD117, CD66b, CXCR4, CD34, arginase1 (ARG1), iNOS and PD-L1 conjugated with different fluorescent dyes were purchased from BD Pharmingen (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). The oxidation-sensitive dye CM-H2DCFDA was purchased from Invitrogen (Grand Island, NY).
The expression of markers on the MDSCs and tumor cells was investigated using FACS analysis after surface staining or intracellular staining with human-specific Abs conjugated with different fluorescent dyes. The MDSC phenotype analysis involved gating within the HLA-DR⁻ cell population that expressed both the CD33 and CD11b antigens. Intracellular staining to detect Foxp3 was performed on T cells from PBMCs, tumorous tissues, and paracancerous tissues. After washing, the cells were stained with anti-CD4, fixed, permeabilized with Perm/Fix solution (eBioscience), and stained intracellularly with anti-Foxp3. The cells showing positive staining were detected using the Cytomics FC 500 MPL flow cytometry system (Beckman Coulter) and analyzed with CXP software (Beckman Coulter, Inc. Fullerton, CA, USA).

To measure the levels of YFP-tagged proteasomal substrates, cells were analyzed by the Cytomics FC 500MPL Flow Cytometry System (Beckman Coulter, Brea, CA) following a previously described procedure (Wang et al., 2011 (link)). For the immunofluorescence imaging studies, cells transiently expressing YFP-tagged TCRα were grown in a Lab-Tek slide chamber (Nalge Nunc International, Rochester, NY) were fixed for 20 min in phosphate-buffered saline (PBS) containing 4% paraformaldehyde. Images were taken using an Axiovert 200 inverted microscope with a 63× oil objective lens (Zeiss, Germany). For immunostaining experiments, fixed cells were permeabilized in PBS containing 0.1% NP40 and 5% normal donkey serum and then stained in the same buffer with the indicated antibodies.