Flow cytometry staining was performed as previously described [31 (link)]. To block non-specific staining of surface antigens, which is essential for the detection of microglia/macrophage antigens, 0.5 μL of mouse FcR Blocking Reagent (1:100 dilution; Miltenyi Biotec, Pozuelo de Alarcón, Spain, 130-059-901) was added to blocking buffer containing the cell suspension and incubated for 10 min. Subsequently, the mix containing fluorochrome-conjugated antibodies was added (Table 2 ). Cells were kept at 4 °C in the dark, for 15 min; and then washed with 1 mL of blocking buffer and centrifugated for 5 min at 300× g. Finally, the cell pellets were resuspended in 500 μL of PBS and samples were immediately used for flow cytometry. Data were acquired using a MACSQuant flow cytometer and analyzed with MACSQuantify software (Miltenyi Biotec). Prior to sample analysis, fluorescent calibration beads (1000 beads/µL) were added to count the absolute number of cells. After excluding cellular debris and aggregates, a minimum of 10,000 events corresponding to individual viable cells were examined in each analysis. The cell number measurements are presented as normalized with respect to their controls.
Mouse fcr blocking reagent
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The Mouse FcR Blocking Reagent is a laboratory product designed to block Fc receptors on mouse cells. It is intended to prevent non-specific binding of antibodies during immunoassays and flow cytometry experiments involving mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (5 mentions)
Facscanto 2, by BD (3 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (3 mentions)
Streptavidin energy coupled dye, by Beckman Coulter (3 mentions)
Dispase 2, by Roche (3 mentions)
Facscalibur, by BD (3 mentions)
Influx cell sorter, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Macsquantify software, by Miltenyi Biotec (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Roche (2 mentions)
Collagenase p, by Roche (2 mentions)
Cd3e fitc, by Thermo Fisher Scientific (2 mentions)
Ifn γ pe, by BioLegend (2 mentions)
Facs staining buffer, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ec800 cell analyzer, by Sony (2 mentions)
Gentlemacs c tube, by Miltenyi Biotec (2 mentions)
Fluorescence labeled isotype matched control antibodies, by BioLegend (2 mentions)
Facsverse, by BD (2 mentions)
50 protocols using mouse fcr blocking reagent
1
Flow Cytometry Staining Protocol for Microglia
2
Multicolor Flow Cytometry of Cell Surface Markers
3
Multiparameter Flow Cytometry Analysis of Immune Cell Subsets
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Samples were blocked with mouse FcR Blocking Reagent (MiltenyiBiotec, USA), and then stained with the indicated antibodies. The antibodies included APC/Cy7 anti-interferon (IFN)γ, APC/Cy7 anti-CD38, BV421 anti-CCR7, BV510 anti-CXCR3, PerCP/Cy5.5 anti-CXCR5, biotinylated anti-CXCR5 from Biolegend (USA); AF488 anti-CD4, eFluor506 anti-IL17A, PE anti-IL21, PE-Cy7 anti-CD44, PE-Cy7 anti-FoxP3 from eBiosciences (USA); FITC anti-CD69, PE anti-CD25, PerCP anti-CD3e, PE-Cy7 anti-IL4 from BD Biosciences (USA); biotinylated anti-PD-1 from R&D systems (USA) and streptavidin-APC from Jackson Immunoresearch (USA). When necessary, red blood cell lysis was performed using BD Pharm Lyse lysing buffer (BD Biosciences). For intracellular staining, CytoFix/Perm kit (BD Biosciences) or FoxP3/Transcription Factor Staining Buffer Set (eBiosciences) were used according to the manufacturer’s instructions. Cytometric data were collected using an FACS Canto II (BD Biosciences) and analyzed using FlowJo software (BD Biosciences). Gating strategy is shown in online supplemental figure 4 .
4
Phenotypic Analysis of Murine BALF Cells
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BALF cells were suspended in mouse FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min prior to staining with FITC-, PE-, biotin-, PerCP/Cy5.5-, or PC7-conjugated antibodies. After staining, biotinylated antibodies were visualized with streptavidin-energy-coupled dye (Beckman Coulter, Fullerton, CA). The antibodies used were anti-F4/80, anti-CD19, anti-Ly-6G, anti-NK1.1, anti-CD45, and anti-CD3ε. Stained cells were analysed with a Cytomics FC500 flow cytometer (Beckman Coulter) and Flowjo software (Tree Star, Ashland, OR).
5
Flow Cytometric Analysis of Foxp3+ T Cells
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To analyze cell surface markers by flow cytometry, LP cells were stained for 30 min at 4°C with PE-conjugated anti-mouse CD4 (clone RM4-5; BD Biosciences). Staining for intracellular Foxp3 was performed according to the manufacturer's instructions using a Foxp3 Staining Buffer Set (eBioscience) and APC-conjugated anti-mouse/rat Foxp3 (clone FJK-16s; eBioscience). Cells were then washed in PBS with 1% bovine serum albumin (BSA) and permeabilized for 1 h in Fixation/Permeabilization working solution (eBioscience). Cells were pre-incubated with a mouse FcR blocking reagent (Milteny Biotec, Auburn, CA, USA) for 5 min before staining to prevent antibody binding to the Fc receptor. Staining with isotype control antibodies was performed in all experiments, and the discrimination of positively stained cells was based on data obtained with isotype control antibodies. Flow cytometry analysis was performed using a FACS Canto II (BD Biosciences).
6
Tumor Cell Population Analysis
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For cell population analysis, tumors were minced in small pieces and incubated with an enzymatic cocktail containing collagenase P (200 µg/mL; Roche), dispase II (800 µg/mL; Roche) and DNase I (100 µg/mL; Roche) in DMEM at 37°C, with vortex and mixing every 20 min 3-4 times. Disaggregated tissue was filtered using a 40 µm cell strainer and centrifuged at 16 100 g for 7 min. Cells were resuspended in FACS buffer (0.05% sodium azide, 0.5% bovine serum albumin and PBS) and stained with the viability dye Zombie Aqua (Biolegend), for 20 min in the dark at RT. After washing, cells were incubated with mouse FcR blocking reagent (Milteny Biotec) for 10 min in the dark at RT. Then, the antibodies described in Supplementary Table S3 were added and incubated for 30 min at 4°C. After washing, cells were fixed with 150 µL of BD Cytofix/Cytoperm™ (BD Biosciences) and acquired in the next day in a BD LSR Fortessa flow cytometer. FlowJoTM 10.8.1 software (BD Biosciences) and OMIQ (Dotmatics, Boston, Massachusetts, USA) were used for data analysis.
7
Flow Cytometry Analysis of Immune Cells
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Single-cell suspensions were blocked with mouse FcR blocking reagent (Miltenyi Biotec) for 10 min at 4 °C prior to surface staining. Cell viability was assessed by Fixable viability dye eFluor 520 (eBioscience) to exclude dead cells. The following anti-mouse antibodies were used: FITC-CD11b, PE-F4/80, APC-F4/80, FITC-CD45, PerCP-eFluor 710-MHC Class II (I-A/I-E), PerCP-eFluor 710-CD3, FITC-CD3e, FITC-CD4, APC-CD4, APC-CD8a, PE-PD-L2, PE-B7-H2, PE-B7-H3, PE-B7-H4, PE-TIM-4, PE-VISTA from eBioscience; APC-CD206, PE-PD-L1, APC-CD86, PE/Cy7 Ki-67 from Biolegend; V450-CD4, BV510-CD8, PE-IFN-γ from BD. The following anti-human antibodies were used: PerCP-eFluor 710-CD3, APC-CD4, APC-CD8a from eBioscience; FITC-CD14, PE-PD-L1, APC-CD163, APC-CD86, FITC-HLA-DR, PE-Cy7-CD163, PE-CD25, PE-IFN-γ from Biolegend; FITC-CD8, PE-IL-10 from BD. For intracellular staining, cells were fixed and permeabilized with the Fixation/Permeabilization solution kit (BD). All flow cytometry data was acquired on FACSCalibur or LSRFortessa (BD, San Jose, USA) and analyzed by FlowJo V10 (TreeStar, Ashland, USA).
8
Detecting NK Cell Stimulatory Ligands on Stem Cells
9
Multicolor Flow Cytometry Immunophenotyping
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FACS Staining Buffer (Thermofisher Scientific, Waltham MA, USA) was used to stain cells on ice. FC receptors (FcR) were blocked using mouse FcR blocking reagent (Miltenyi Biotec, Auburn, CA, USA) for 15 minutes protected from light followed by incubation with conjugated antibodies on ice for 30 minutes. Cells were then washed and re-suspended in FACS buffer and data was acquired using MACSQuant analyzer machine and MACSQuantify software (Miltenyi Biotech Auburn, CA, USA). Antibodies used: CD11c APC; clone N418 (Affymetrix, eBioscience, Thermofisher Scientific, Waltham MA, USA)., CD86 (B7-2) PE; clone GL1(Affymetrix, eBioscience, Thermofisher Scientific, Waltham MA, USA)., CD80 FITC; clone 16-10A1(Invitrogen, Thermofisher Scientific, Waltham MA, USA), CD4 Vioblue; clone GK1.5 (Invitrogen, Thermofisher Scientific, Waltham MA, USA), MHCII Viogreen; clone M5/114.15.2, (Miltenyi Biotech Auburn, CA, USA).
10
Tumor Immune Profiling by CyTOF
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Mouse tumor samples were processed as previously described to obtain single-cell suspensions (Liu et al., 2018). Tumor-infiltrating immune cells were enriched in a Ficoll gradient as described previously (Liu et al., 2018). Cells were washed with CyTOF PBS and stained with cisplatin solution (1:2000, Fluidigm Cat#201064) for 2 min. Then, cells were washed twice with CyTOF PBS and stained with mouse FcR-Blocking reagent (1:100, Miltenyi Biotec Cat#130-092-575) at RT for 10 min followed by staining with metal-coupled extracellular antibodies cocktail (Supplementary Table 1 ) at RT for 30 min. Cells were washed twice and resuspended in 2% formaldehyde diluted from 16% formaldehyde (Electron Microscopy Cat#15710) and kept at 4 °C overnight until acquisition day. Prior to the acquisition, cells were stained with Intercalator (1:2000, Fluidigm Cat#201103B) in CyTOF PBS for 10 min and washed once with CyTOF PBS, once with milliQH20 and resuspended in Maxpar Cell Acquisition Solution (Fluidigm, Cat#201237) and analyzed using the CyTOF 3 Helios system (Fluidigm, instrument located at the Dana Farber Cancer Institute Mass Cytometry Core). Data were processed using Cytobank.
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