Freshly isolated monocytes and adhered monocytes with or without GM-CSF (100ng/ml) or M-CSF (100ng/ml) treatment were immunostained with appropriate antibodies using conventional surface and/or intracellular staining methods. Cells were stained intracellularly using the BD Cytofix/Cytoperm Buffer kit (BD Biosciences, San Jose CA) according to manufacturer’s instructions. Intracellular active β-catenin was measured using a primary monoclonal β-catenin antibody that detects the active form of β-catenin that is hypophosphorylated on serine 37 and threonine 41 (US Biological, MA) and a secondary goat F (ab’2) anti-mouse (CALTAG, CA) antibody. Intracellular HIV-1 Core Antigen was detected using a monoclonal antibody (Beckman Coulter Brea, CA). CD3, CD4, CD14, CD16, CD80, CD86 and HLA-DR surface stains were detected using antibodies from BD Biosciences (Franklin Lakes, NJ). LSR II flow cytometer with FACSDiva software (BD) was used for data collection in Figure 2 . BDFACSVerse flow cytometer with BD FACSuite software (BD) was used for data collection in figures 1 and 5 . FlowJo software (Tree Star; Ashland, OR) was used for analysis. Side scatter area (SSC-A) versus Aqua Live/Dead staining and forward scatter area (FSC-A) versus forward scatter height (FSC-H) were employed to exclude dead cells and doublets, respectively.
Cytofix cytoperm buffer kit
Manufactured by BD
The BD Cytofix/Cytoperm Buffer kit is a laboratory reagent used for the fixation and permeabilization of cells prior to intracellular staining and flow cytometric analysis. The kit contains a fixation buffer and a permeabilization/wash buffer.
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4 protocols using cytofix cytoperm buffer kit
1
Flow Cytometry Analysis of Monocyte Activation
2
Cytokine Production Assay in Spleen Cells
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After isolation of spleen cells, 2x106 cells were seeded in a 96-well round-bottom plate and incubated for 5 h with 1 mg/ml Phorbol-myristate-acetate (PMA) and 0.25 mg/ml Ionomycin (Iono) in 200 µl complete RMPI 1640 medium (RPMI 1640 + 5% FCS + 2 mM L-glutamine + 10 mM HEPES + 50 µg/ml Gentamicin) to induce cytokine production. To accumulate cytokines within the cells the medium was also supplemented with Brefeldin A and Monensin (BioLegend) according to the manufacturer’s instructions for the whole incubation period. αCD107a APC antibody (clone 1D4B, RRID: AB_2234505, BioLegend) was added 1:100 to the medium to improve the CD107a staining. BD Cytofix/Cytoperm buffer kit (BD Biosciences) was used for fixation and intracellular staining according to the manufacturers instructions for the restimulation assay.
3
Flow Cytometric Analysis of MM and pDCs
4
Dissociation and Characterization of Tumor-Infiltrating Lymphocytes
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For detecting the TILs, the tumors were cut into small pieces, mechanically disrupted, and filtered through a 70-μm mesh to generate a single-cell suspension58 . For cell-surface staining, cell suspensions were washed twice in PBS and stained with indicated fluorescent-labeled antibodies for 30 min on ice and washed with PBS. For intracellular staining, the cells were sorted for fixation and permeabilization using the Cytofix/CytoPerm buffer kit (Cat# 554714, BD Bioscience). For detecting the proliferation and activation of T cell, human T cells were labeled with CFSE, and activated in vitro with anti-CD3 pre-coated U-96-plates in the presence of 100 U/ml IL2. When needed, irradiated (100 Gy) breast cancer cells were added to coculture system at the indicated ratio in triple. All flow cytometry analysis was conducted on CytoFlex (Beckman) or Gallios (Beckman), and the data were analyzed using FlowJo software (FlowJo Vx.0.7), Kaluza Analysis software (Kaluza Analysis Version 2.1), or CytExpert software (CytExpert 2.4) according to manufacturers’ instructions. Compensation beads were used to evaluate spectral overlap, compensation was automatically calculated. Antibodies for flow cytometry analysis were used at a dilution of 1:20–1:50. All antibodies used for flow cytometry analysis were listed in Supplementary Table 5 .
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