Analyses were performed using the following antibodies: CD3-FITC (145-2C11, Biolegend), CD4-BV750 (GK1.5, Biolegend), CD8-BV786 (53-6.7, Biolegend), CD62L-BV510 (MEL-14, Biolegend), CD44-PE/Cy5 (IM7, Biolegend), CD107a-BV711 (1D4B, Biolegend), CD154-PE (MR1, BD), Gmzb-AF700 (QA16A02, Biolegend), IFN-γ-APC (XMG1.2, Biolegend) and TNF-α-BB700 (MP6-XT22, BD). In brief, splenocytes isolated from experimental mice were cultured in the presence of phorbol 12-myristate 13-acetate (PMA) (1.9 nM) and ionomycin (0.08 mg/ml) solution at 37°C for 4 h. After washing, 2 × 106 cells were stained with FVS780 (viability dye) followed by treatment with antibodies for surface antigens (Anti-CD3, CD4, CD8, CD62L, CD44, CD107a, and CD154) at 4°C for 30 min. After washing, cells were fixed and permeabilized with BD Cytofix/Cytoperm, and then stained with the antibodies against the intracellular antigens (Gzmb, IFN-γ, and TNF-α) for 20 min at 22°C. Stained cells were separated using a BD FACSymphony™ Cell Analyzer and one million events were collected for each sample. The analysis was performed in FlowJo software version 10.6.2.
Facsymphony cell analyzer
Manufactured by BD
The BD FACSymphony is a highly advanced cell analyzer designed for multiparametric analysis. It utilizes flow cytometry technology to precisely measure and characterize a wide range of cell populations. The instrument is equipped with multiple lasers and detectors, enabling the simultaneous detection and quantification of diverse cellular properties.
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Lab products found in correlation
Dnase 1, by Roche (3 mentions)
Facsaria, by BD (3 mentions)
Cd45 microbeads non human primate, by Miltenyi Biotec (2 mentions)
Ifn γ apc xmg1, by BioLegend (1 mentions)
Cd62l bv510 mel 14, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (1 mentions)
6 protocols using facsymphony cell analyzer
1
Multiparametric T Cell Analysis
2
Isolation of Rhesus Brain-Derived CD45+ Cells
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Cryopreserved mononuclear cells from rhesus brain were thawed at room temperature, placed in fresh complete media (For splenic cells: RPMI supplemented with 10% HI-FBS, 1% L-glutamine, 1% penicillin-streptomycin; For brain tissue derived cells: DMEM supplemented with 10% HI-FBS, 1% L-glutamine, 1% penicillin-streptomycin) and treated with 2 units/mL of DNase I (Roche Diagnostics) for 15 minutes at 37°C. Cells were washed in complete media and CD45+ cells isolated using CD45 magnetic bead separation for non-human primates (Miltenyi Biotec CD45 Microbeads non-human primate) in accordance with the manufacturer’s protocol. Enriched CD45+ cells were stained for CD45 and a live dead marker for subsequent flow cytometric sorting. Live CD45+ cells were characterized and quantified on a BD FACSymphony cell analyzer and sorted utilizing a FACS Aria and suspended in RPMI for single cell RNA sequencing studies.
3
Cell Staining, Sorting, and Analysis
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Cell staining and sorting were performed as previously described (12 (link)). Fluorescence was measured using a BD FACSymphony cell analyzer with FACSDiva version 8.0.1 software. Compensation, gating, and analysis were performed using FlowJo (versions 9 and 10). Cell sorting was performed using a BD FACSAria III cell sorter. Reagents used for flow cytometry are listed in Table 1 .
4
Isolating CD45+ Cells from Cryopreserved Rhesus Tissues
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Cryopreserved mononuclear cells from rhesus brain and splenic tissues were thawed at room temperature, placed in fresh complete media (For splenic cells, RPMI supplemented with 10% HI-FBS, 1% L-glutamine, 1% penicillin-streptomycin; For brain tissue derived cells, DMEM supplemented with 10% HI-FBS, 1% L-glutamine, 1% penicillin-streptomycin [Sigma-Aldrich]) and treated with 2 units/mL of DNase I (Roche Diagnostics) for 15 minutes at 37°C. Cells were washed in complete medium and CD45+ cells were isolated using CD45 microbeads for nonhuman primates (Miltenyi Biotech) in accordance with the manufacturer’s protocol. Enriched CD45+ cells were stained for CD45 and a live-dead marker for subsequent flow cytometric sorting. Live CD45+ cells were characterized and quantified on a BD FACSymphony cell analyzer, sorted utilizing a FACS Ari,a and suspended in DMEM for scRNA-Seq studies.
5
Lipid Peroxidation Measurement by Flow Cytometry
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Lipid peroxidation was assessed by flow cytometry with BODIPY™ 581/591 C11 (Thermo Fisher Scientific, United States) following the manufacturer’s instructions. In brief, cells were pretreated with 1 µM BSO or 20 µM Fer-1. Cells were collected at 48 h for lipid peroxidation and to measure cell viability. Cells were resuspended in 400 μl of serum-free medium that contained BODIPY™ 581/591 C11 (5 µM) at 37 °C in the dark for 30 min, to detect lipid peroxidation. Finally, cells were stained with Ghost Dye Red 780 (Tonbo Biosciences) and analyzed by flow cytometry immediately. FeRhoNox (Gorya Chemical) was used following manufacturer’s instructions. Briefly, cells were plated overnight and incubated with FeRhoNox in HBSS for 1 h then immediately analyzed by flow cytometry. Data were collected using a BD FACSymphony cell analyzer, and a minimum of 10,000 cells were analyzed per condition. Cells were gated to exclude debris and doublets using FlowJo software and statistical analysis was conducted as described below.
6
Enrichment of brain-derived CD45+ cells
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Cryopreserved mononuclear cells from rhesus brain were thawed, placed in fresh complete media (For splenic cells: RPMI supplemented with 10% HI-FBS, 1% L-glutamine, 1% penicillin-streptomycin; For brain tissue derived cells: DMEM supplemented with 10% HI-FBS, 1% L-glutamine, 1% penicillin-streptomycin) and treated with 2 units/mL of DNase I (Roche Diagnostics) for 15 minutes at 37°C. Cells were washed in complete media and CD45+ cells isolated using CD45 magnetic bead separation for non-human primates (Miltenyi Biotec CD45 Microbeads non-human primate) in accordance with the manufacturer’s protocol. Enriched CD45+ cells were stained for CD45 and a live dead marker for subsequent flow cytometric sorting. Live CD45+ cells were characterized and quantified on a BD FACSymphony cell analyzer and sorted utilizing a FACS Aria and suspended in RPMI for single cell RNA sequencing studies.
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