Patients diagnosed with relapsing remitting MS (n = 18) based on the revised McDonald Diagnostic Criteria were recruited from the Multiple Sclerosis Clinic at the University of Sherbrooke by a board-certified neurologist. On the day of blood sampling, all subjects were afebrile and had no signs and symptoms of infection based on history, physical examination, and responses to a survey. The age-matched control group consisted of 23 healthy volunteers. The group’s demographic and clinical data are shown in S2 Table . PBMCs were separated by ficoll and RNA extraction was done using Trizol. The CD3+ T cells and CD14+ monocytes were sorted from frozen PBMCs using BD FACSAriaII (70-μm nozzle) at Johns Hopkins Sidney Kimmel Cancer Center Flow Cytometry and Immune Monitoring Core facility. Cells were stained with CD14 FITC and CD3 APCVio770 (Miltenyi). Cell viability was determined using Helix NP Blue (Biolegend). The expression of NLRX1 was quantified using qPCR and normalized to the internal control GAPDH.
Cd3 apc vio770
Manufactured by Miltenyi Biotec
Sourced in Germany
CD3-APC-Vio770 is a fluorochrome-conjugated monoclonal antibody that binds to the CD3 antigen expressed on the surface of T cells. It is designed for use in flow cytometry applications to identify and quantify T cell populations within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cd62l vioblue, by Miltenyi Biotec (3 mentions)
Cd16 pe, by Miltenyi Biotec (3 mentions)
Cd8 viogreen, by Miltenyi Biotec (3 mentions)
Cd4 vioblue, by Miltenyi Biotec (3 mentions)
Cd20 pe vio770, by Miltenyi Biotec (2 mentions)
Cd45 vioblue, by Miltenyi Biotec (2 mentions)
Cd45ra pe, by Miltenyi Biotec (2 mentions)
Cd45ro apc, by Miltenyi Biotec (2 mentions)
Cd56 pe vio770, by Miltenyi Biotec (2 mentions)
Ccr7 fitc, by Miltenyi Biotec (2 mentions)
Cd19 fitc, by Miltenyi Biotec (2 mentions)
Cd11c pe, by Miltenyi Biotec (2 mentions)
Helix np blue, by BioLegend (1 mentions)
Cd4 alexa fluor 700, by Thermo Fisher Scientific (1 mentions)
Ifn γ efluor450, by Thermo Fisher Scientific (1 mentions)
Il2 percpefluor710, by Thermo Fisher Scientific (1 mentions)
Foxp3 alexa fluor 488, by BioLegend (1 mentions)
Cd127 pe, by Miltenyi Biotec (1 mentions)
Foxp3 buffer set, by Thermo Fisher Scientific (1 mentions)
Streptavidin alexa fluor 700, by Thermo Fisher Scientific (1 mentions)
10 protocols using cd3 apc vio770
1
Profiling NLRX1 Expression in MS
2
Comprehensive Immune Phenotyping Protocol
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All staining experiments were performed at 4°C for 30 minutes. Antibodies used were CD3-PerCPCy5.5, CD3-AF700, CD4-Brilliant Violet 605, CD8-APC-Cy7, CD25-APC, CD134-PE, OX40-APC, TNF-α-PECy7, CD154-APC, CD152-PCF594, Tbet-PerCPCy5.5, PD1-PeCy7, CD15s-Brilliant Violet 421, Helios-PE, ICOS-PeCy7, CD39-Brilliant Violet 711 (Becton Dickinson (BD) Biosciences), CD4-Alexa Fluor 700, IFN-γ-eFluor450, IL2-PerCPeFluor710, Streptavidin-Alexa Fluor 700 (eBioscience), FoxP3-Alexa Fluor 488, CD25-Brilliant Violet 421 (BioLegend), CD39-biotin, CD127-PE, CD4-APCVio770, CD3-APCVio770 (Miltenyi biotec), Streptavidin-ECD, CD45RO-ECD (Beckman Coulter). LIVE/DEAD fixable aqua staining kit (Life technologies) was used to discriminate live and dead cells. For intracellular staining, FoxP3 buffer set (eBioscience) was used. Cell acquisition was performed by an LSR II (Becton Dickinson) and analyses were performed using FlowJo software.
3
Multicolor Flow Cytometry of Whole Blood
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We used CD3-APC-Vio770, CD4-PE, CD8-PE-Vio770, and CD45RO from Miltenyi Biotec (Germany) and CD197-CCR7-FITC from BD Biosciences (Belgium) to stain the whole blood for 30 minutes. A 6-color BD FacsVerse instrument (BD Biosciences) was used to conduct the flow cytometry, and Flow Jo (version 10) was used to analyze the data. The gating strategies are shown in Figure S2 .
4
Immune Cell Profiles in Pregnancy and Multiple Sclerosis
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CD4+ and CD8+ T lymphocytes, Treg, NKbright and M2 monocytes were evaluated in blood specimen of HC and MS patients at different trimester of pregnancy. In addition, the same populations were analyzed in decidual tissues. The analyses were performed by a biologist blinded to the clinical data.
Non-specific sites of 3 × 106 cells were blocked with rabbit immunoglobulins G (IgG, Sigma-Aldrich), and cells were then incubated with fluorochrome-conjugated monoclonal Ab (mAb) and isotype–matched negative controls for 10 min at 4°C. The following anti-human mAbs were used: CD163 Phycoerythrin (PE) and CD14 Fluorescein isothiocyanate (FITC) for M2 monocytes, CD3 allophycocyanin (APC)-Vio770, CD16 FITC and CD56 APC for NK cells, CD3 APC-Vio770, CD4 PE-Vio770 and CD8 FITC for T cells, CD4 PE-Vio770, CD25 APC, and CD127 FITC for Treg (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA). The gating strategy is shown in theFigure 1 .
Non-specific sites of 3 × 106 cells were blocked with rabbit immunoglobulins G (IgG, Sigma-Aldrich), and cells were then incubated with fluorochrome-conjugated monoclonal Ab (mAb) and isotype–matched negative controls for 10 min at 4°C. The following anti-human mAbs were used: CD163 Phycoerythrin (PE) and CD14 Fluorescein isothiocyanate (FITC) for M2 monocytes, CD3 allophycocyanin (APC)-Vio770, CD16 FITC and CD56 APC for NK cells, CD3 APC-Vio770, CD4 PE-Vio770 and CD8 FITC for T cells, CD4 PE-Vio770, CD25 APC, and CD127 FITC for Treg (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA). The gating strategy is shown in the
5
Flow Cytometry Immunophenotyping Panel
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MACSQuant Analyzer, MACSQuant Analyzer VYB (Miltenyi Biotec), or FACSCanto, LSRII (Becton Dickinson, NJ) was used to analyze cell populations by flow cytometry. The following antibody and protein conjugates were used: CD3-PE, CD3-APC-Vio770, CD4-APC, CD4-VioGreen, CD8-APC-Vio770, CD8-VioGreen, CD14-APC, CD16-PE, CD19-APC, CD20-PerCP-Vio770, CD20-PE-Vio770, CD34-APC, CD34-PE, CD45-VioBlue, CD45-VioGreen, CD45RO-PE-Vio770, CD56-PE, CD62L-VioBlue, CD95-APC, CD45-VioBlue (mouse) (all from Miltenyi Biotec); ErbB2-Fc fusion protein (R&D Systems), anti-human-IgG (Fc gamma-specific) PE (eBioscience). 7AAD staining was used for dead cell exclusion and Violet Cell Trace (Thermo Fisher) for SupT1 cell tracking.
6
Immunophenotyping of PBMC and T-cells
7
Flow Cytometry Analysis of Immune Cells
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The differential immune cell count was determined by using either NovoCyte or Beckman Coulter CytoFLEX flow cytometers and analyzed with NovoExpress (Acea Biosciences, USA) or CytExpert software (Beckman Coulter, USA). Differential immune cell counts were performed as previously described (Chan et al. 2016 ). Leukocytes were identified as CD45+, alveolar macrophages as CD11c+Siglec-F+, eosinophils as CD11c−Siglec-F+, neutrophils as GR-1+CD11b+ or Ly-6G+CD11b+, B cells as CD3−/CD19+ and T cells as CD3+/CD19− cells. All antibodies were from Miltenyi Biotech: CD45-VioBlue (130–110-802), CD45-PE (130–110-797), CD64-APC-Vio770 (130–118-685), Siglec-F-PE-Vio770 (130–112-334), Siglec-F-APC (130–102-241), CD11b-PE-Vio615 (130–113-807), CD11b-FITC (130–098-085), CD11c-PE-PerCP700 (130–110-842), CD11c-PE (130–102-799), Ly-6G-PerCP700 (130–117-500), Gr-1-PE (130–112-306), Gr-1-APC (130–102-833), CD3-APC-Vio770 (130–119-793), CD19-PE-Vio770 (130–111-885), Annexin-V-PE (130–118-363), and Viobility 405/520 Fixable Dye (130–109-814).
8
Comprehensive CBMC and T Cell Immunophenotyping
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Staining of cell surface markers on CBMCs and T cells was performed with CD3-APC/Vio770, CD4-Vioblue, CD8-Viogreen, CD19-FITC, CD56-PE/Vio770, CD16-PE, CD62L-Vioblue, CD45RA-PE, CD45RO-APC, and CCR7-FITC (Miltenyi Biotec and BD Biosciences). 50,000 events per sample were acquired on a MACSQuant cytometer (Miltenyi), and the data were analyzed with Flow Jo (Tree Star).
9
Multicolor Flow Cytometry for Immune Cell Phenotyping
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Flow cytometric analyses of frequencies of activated B cells, T cells, and monocytes were performed using EDTA anticoagulated blood predose and at 24 h postdose. The following antibody cocktail was used for B and T cell phenotyping: CD3 APC-Vio770 (130-104-236; Miltenyi), CD4 PE (550630; BD Biosciences), CD8 PerCp-Vio700 (130-097-911; Miltenyi), CD20 PE-Vio770 (130-099-733; Miltenyi), CD69 VioBlue (130-106-596; Miltenyi), CD25 BB15 (565096; BD Biosciences), and Live/Dead Aqua (1737195; Molecular Probes). The following antibody cocktail was used for monocyte phenotyping: CD14 PE-Cy7 (130-091-242; Miltenyi), CD16 APC Vio770 (130-106-765; Miltenyi), and CD169-APC (346008; BD Biosciences). Data (150,000 events) were acquired on a MACSQuant analyzer 10 and analyzed by FlowJo software.
10
Multicolor Flow Cytometry of Immune Cells
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Antibody reagents were obtained from Miltenyi Biotech: REA clones of antimouse CD3-APC-Vio770 (REA606), CD4-Vioblue (REA 604), CD8a-PE-Vio770 (REA 601), Foxp3-Vio515 (REA 788), NKp46-APC (REA 815), CD11c-PE (REA 754), CD11b-APC-Vio770 (REA 592), Ly6C-Vioblue (REA 796), Ly6G-PE-Vio770 (REA 526). Foxp3 staining was performed using Foxp3 Staining Buffer Set (Miltenyi Biotech).
Flow cytometry analysis was performed using a Miltenyi 8-color MACSQuant, and data were analyzed using Flowlogic (Miltenyi Biotech).
Flow cytometry analysis was performed using a Miltenyi 8-color MACSQuant, and data were analyzed using Flowlogic (Miltenyi Biotech).
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