The following anti-human antibodies were used for staining: CD3-FITC, CD3- Alexa Fluor 700, CD8a-Alexa Fluor700, CD4-Percp-Cy5.5, CD4-Alexa Fluor 700, CD25-PE, CD25-PE-Cy7, Foxp3-FITC, Foxp3- Percp-Cy5.5, Foxp3-BV421, CD127-APC, CD39-APC, LAP-PE, IL-10-PE, TIM-3-PE, TIM-3-BV421, IFN-γ receptor-PE, IFN-γ- APC, Granzyme B-PE-dazzle E, PD-1-APC, PD-1- PE-Cy7, PD-1- Percp-Cy5.5, CTLA-4-PE, CTLA-4-FITC, ki67-Alexa Fluor 488 and their respective isotype controls were purchased from Biolegend. Recombinant human IFN-γ (R&D Systems) was used at 200 ng/ml. Anti-PD-1 Ab (Nivolumab from Bristol-Myers Squibb), anti-Tim-3 (clone 2E2 from Biolegend) and isotype were used at 10µg/ml.
Cd4 alexa fluor 700
Manufactured by BioLegend
Sourced in United States
CD4-Alexa Fluor 700 is a fluorophore-conjugated antibody that binds to the CD4 protein on the surface of T cells. It is designed for use in flow cytometry applications to identify and quantify CD4+ T cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BioLegend (3 mentions)
Lsrfortessa, by BD (3 mentions)
Cd3 pacific blue, by BioLegend (2 mentions)
Il 17a pe, by BioLegend (2 mentions)
Lsr 2 cytometer, by BD (2 mentions)
Cd4 percp cy5.5 rpa t4, by BioLegend (2 mentions)
Hiv p24 fitc kc57, by Beckman Coulter (2 mentions)
Hiv p24 pe kc57, by Beckman Coulter (2 mentions)
Ccr6 pe, by BioLegend (2 mentions)
Cd45ra alexa efluor 780, by BioLegend (2 mentions)
Rorc2 alexa fluor 647, by BioLegend (2 mentions)
Ifn γ alexa fluor 700, by BioLegend (2 mentions)
Cd45ra alexa efluor 780, by BD (2 mentions)
Cd3 pacific blue, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Aria 2, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Cd3 apc h7, by BD (2 mentions)
15 protocols using cd4 alexa fluor 700
1
Comprehensive Immune Phenotyping Protocol
2
Multiparametric Flow Cytometry of Murine Thymocytes
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Thymocyte cell suspensions were stained for 20 min at room temperature with CD4-APC, CD8a-PE, TCRb-BV421 and CD69-FITC (BD-Pharmingen). Single cells were sorted into 96-well plates containing lysis buffer using a FACSAria Fusion flow cytometer (BD Biosciences) and the gates depicted in Supplementary Fig. 1 . Flow cytometry standard files were analysed with DIVA (BD Biosciences) and FlowJo v10 (TreeStar Inc) analysis software.
C57BL/6 (C57BL/6OlaHsd, Envigo, UK) and Mice lacking MHC class II expression41 (link) (JAX stock #003584) were maintained separately under specific pathogen-free conditions under Project Licences issued by the Home Office, UK. OT-I Rag2−/− and CD8.4 OT-I Rag2−/− mice32 (link) were maintained together at the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, in accordance with laws of the Czech Republic. Six-week-old male or female mice were killed by cervical dislocation, and thymocyte or lymph-node cell suspensions were stained with CD4-APC, TCRb-FITC, CD69-BV421 (Pharmingen) or CD4-Alexa Fluor 700, CD8a-PE or CD8a-BV421, TCRb-APC (Biolegend), and LIVE/DEAD NIR (ThermoFisher). Data acquisition was on a Cytek Aurora flow cytometer (Cytek Biosciences) and flow cytometry standard files were analysed with FlowJo v10 (TreeStar Inc) analysis software. Data were further analysed using GraphPad Prism v5.04 (GraphPad Software).
C57BL/6 (C57BL/6OlaHsd, Envigo, UK) and Mice lacking MHC class II expression41 (link) (JAX stock #003584) were maintained separately under specific pathogen-free conditions under Project Licences issued by the Home Office, UK. OT-I Rag2−/− and CD8.4 OT-I Rag2−/− mice32 (link) were maintained together at the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, in accordance with laws of the Czech Republic. Six-week-old male or female mice were killed by cervical dislocation, and thymocyte or lymph-node cell suspensions were stained with CD4-APC, TCRb-FITC, CD69-BV421 (Pharmingen) or CD4-Alexa Fluor 700, CD8a-PE or CD8a-BV421, TCRb-APC (Biolegend), and LIVE/DEAD NIR (ThermoFisher). Data acquisition was on a Cytek Aurora flow cytometer (Cytek Biosciences) and flow cytometry standard files were analysed with FlowJo v10 (TreeStar Inc) analysis software. Data were further analysed using GraphPad Prism v5.04 (GraphPad Software).
3
Characterization of Th17 Cell Subsets
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The following fluorochrome-conjugated Abs were used for flow cytometry analysis: HIV-p24 FITC (KC57) (Beckman Coulter), HIV-p24 PE (KC57) (Beckman Coulter), CD3 Pacific blue (UCHT1), CD4 PerCP/Cy5.5 (RPA-T4) (BioLegend), CD4 Alexa Fluor 700 (RPA-T4), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), RORC2 Alexa Fluor 647 (Q31-378), Ki-67 BUV395 (B56), IL-17A PE (eBio64DEC17), and IFN-γ Alexa Fluor 700 (B27). The Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid, Life Technologies) was used to exclude dead cells. Intracellular staining was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences), and intranuclear staining was performed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set. Cells were analyzed with the BD-LSRII cytometer, BD LSRFortessa and BD-Diva (BD Biosciences), and FlowJo version 10 (Tree Star, Inc.). The positivity gates were placed using fluorescence minus one strategy (8 (link), 20 (link)). For fluorescence-activated cell sorting (FACS), memory CD4 T cells from the PBMCs of ART+ PLWH were isolated by negative selection using magnetic beads. CCR6+RORC2+, CCR6+RORC2−, and CCR6−RORC− T cells were sorted by FACS (BDAria II; BD Biosciences) using the Abs CD3 Pacific blue (UCHT1), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), and RORC2 Alexa Fluor 647 (Q31-378).
4
T Cell Proliferation Assay with Myeloid Cells
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Splenocytes were stained with the PKH26 membrane dye (Sigma-Aldrich) following the manufacturer’s instructions. For the T cell proliferation assay, the PKH26-stained splenocytes were plated at 2.5 × 104/well on a 96-well plate in RPMI supplemented with 10% FBS, 1% (v/v) penicillin/streptomycin solution (Gibco), 1 mM sodium pyruvate, and 50 µM β-mercaptoethanol. Splenocytes were either unstimulated and cultured as a monoculture or splenocytes were stimulated with murine CD3/CD28 Dynabeads (Thermofisher Scientific, Darmstadt, Germany; 1:1 ratio) and 30 U/mL murine rIL-2 (Peprotech) and cultured as a monoculture or as a co-culture with purified CD11b+ Gr+ bone marrow cells in a 1:1 or 1:0.5 cell ratio. The cells were incubated for 3 d at 37 °C with 5% CO2, then subsequently the T cells were assessed for their proliferation rate and cell number by flow cytometry. Cells were stained for CD3-FITC, CD4-Alexa Fluor 700, CD8α-APC antibodies (all Biolegend), and DAPI and collected in TruCount tubes (BD Bioscience). Proliferation was normalized to T cells stimulated with CD3/CD28 Dynabeads and rIL-2, which were set to 100%.
5
SARS-CoV-2 Specific T Cell Profiling
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PBMC were isolated from CPT tubes (BD Biosciences) according to the manufacturer’s instructions and cryopreserved in CTL-CryoTM ABC Media Kit (ImmunoSpot). Before T cell stimulation the PBMC vials were thawed, washed, counted, and suspended in an X-Vivo15 serum-free medium (Lonza). 1-2 million of PBMCs were used per condition. Overlapping peptide pools of SARS-CoV2 spike, nucleocapsid, and membrane proteins were purchased from Miltenyi Biotec and used at a final concentration of 1ug/ml of each peptide. The stimulations (mock, S, M, and N peptide pools) were carried out for 20 hours in the presence of anti-CD28 and anti-CD49d. CEFX peptide pool (JPT Peptides) was used as a positive control. After the stimulation T cells were stained with monoclonal antibodies: CD3 Brilliant Violet 650, CD4 Alexa Fluor 700, CD8 Brilliant Violet 605, CCR7 PE-Dazzle, CD45RA APC, CD69 Brilliant Violet 510 (all from Biolegend), and CD137 PE (from Miltenyi Biotech). Before the acquisition with LSRFortessa (BD Biosciences), 7AAD was added for the discrimination of dead cells. Data were analyzed using FCS Express 7 (DeNovo Software).
6
Comprehensive TRBV Gene Expression Analysis
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Thawed PBMC or DMC were incubated for 30 min at 4°C with live/dead fixable aqua dead cell stain (Life Technologies), CD4 Alexa Fluor 700, CD8 Cy5.5-PerCP, and CD25 Cy7-phycoerythrin (BioLegend). Each sample was then divided and labeled for 30 min at 4°C with 1 of 25 phycoerythrin- or fluorescein isothiocyanate-labeled TRBV-specific mAb (Table A1 in Appendix). Nomenclature used in this study is based on IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org (13 (link)). Functional TRBV genes (14 ) not covered by this panel include; TRBV5-4, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-8, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8, TRBV7-9, TRBV10-1, TRBV10-2, TRBV11-1, TRBV11-3, TRBV12-5, TRBV15, TRBV16, TRBV24-1, and TRBV29-1. After washing with PBS, the FoxP3 Fix/Perm Buffer Set and FoxP3 Alexa Fluor 647 mAb (BioLegend) were used to label intracellular FoxP3, according to the manufacturer’s protocol. Cells were analyzed on a FACSCanto II flow cytometer using FACSDiva software (BD Biosciences). Flow cytometry data were analyzed and presented using FlowJo software (TreeStar). A representative example of cell gating can be found in Data Sheet 2 in Supplementary Material.
7
Multiparameter Immune Profiling of Colorectal Cancer
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Following blocking with FcR blocker, PBMCs, TILs, and NILs were stained with Fixable Viability Dye eFluor® 660 (FVD 660; eBioscience) according to the manufacturer’s protocol to gate for live cells. Cells were then surface labeled with antibodies CD3-APC-H7 (BD Biosciences), CD4-Alexa Fluor 700 (BioLegend), LAP-PE (clone TW4-2F8; BioLegend), and PD-1- PE/Dazzle™ 594 (BioLegend) for 30 min at 4°C. After washing with staining solution, cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience) at 4°C for 45 min after thorough vortexing. Cells were then washed twice with permeabilization wash buffer (eBioscience) and blocked using mouse serum (Sigma-Aldrich) and rat serum (eBioscience) for 10 min. Cells were then stained with antibodies CTLA-4-PerCP-eFluor® 710 (clone 14D3; eBioscience), FoxP3-PE-Cyanine7 (clone PCH101, eBioscience), and Helios-FITC (clone 22F6, BioLegend) for 30 min at 4°C. Cells were washed twice with permeabilization wash buffer (eBioscience) and were resuspended in flow cytometry staining buffer for flow cytometric analyses. Data were acquired with a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Bioscience) and analyzed on BD FACSuite software (BD Biosciences). Out of 12 CRC samples, we did not consider intracellular staining data from 2 samples since no cell viability dye was used for these.
8
Intracellular Cytokine Staining of Tumor Cells
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For intracellular cytokine staining, tumor samples or peripheral blood cells were collected 4 h after the last dose. Tumor samples were homogenized using a gentleMACS dissociator and then filtered through 70 µm cell strainers to prepare a single-cell suspension. Blood cells were lysed with a red blood cell lysis solution. Cells were stimulated with PMA + Ionomycin in the presence of brefeldin A (BD Biosciences; #555029) and monensin (#554724) for 4–6 h. The cells were stained with CD45-BV750 (BioLegend; #103157, clone 30-F11), CD3-APC-Cy7 (BioLegend; #100222, clone 17A2), CD4-Alexa Fluor 700 (BioLegend; #100430, clone GK1.5), and CD8a-Pacific Orange (Thermo Fisher Scientific; #MCD0830, clone 5H10) in the presence of purified rat anti-mouse CD16/CD32 and then fixed and permeabilized using the Foxp3/Transcription Factor staining buffer set. The cells were then stained with IFN-γ-PE (BD Biosciences; #554412, clone XMG1.2) or GranzymeB-Alexa Fluor 647 (BioLegend; #515406, clone GB11). Samples were analyzed using CYTEK Aurora spectral flow cytometry (CYTEK Biosciences, Fermont, CA, USA), and all data were analyzed using FlowJo software.
9
Multiparameter Flow Cytometry Antibody Panel
10
Comprehensive Flow Cytometry Profiling of HIV+ Cells
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The following fluorochrome-conjugated Abs were used for flow cytometry analysis: HIV-p24 FITC (KC57) (Beckman Coulter, Fullerton, CA, USA), HIV-p24 PE (KC57) (Beckman Coulter, Fullerton, CA, USA), CD3 Pacific blue (UCHT1), CD4 PerCP/Cy5.5 (RPA-T4) (Biolegend, San Diego, CA, USA), CD4 Alexa Fluor 700 (RPA-T4), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), RORC2 Alexa Fluor 647 (Q31-378), Ki-67 BUV395 (B56), IL-17A PE (eBio64DEC17) and IFN-γ Alexa Fluor 700 (B27). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid, Life Technologies, Burlington, Ontario, CA) was used to exclude dead cells. Intracellular staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences) and intranuclear staining was performed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set. Cells were analysed with the BD-LSRII cytometer, BD LSRFortessa and BD-Diva (BD Biosciences) and FlowJo version 10 (Tree Star, Inc., Ashland Oregon, USA). The positivity gates were placed using fluorescence minus one (FMO) strategy (8, 20) . For FACS, memory CD4 T cells from PBMCs of ART+ PLWH were isolated by negative selection using magnetic beads. CCR6 + RORC2+, CCR6+RORC2-and CCR6 -RORC-T cells were sorted by FACS (BDAria II; BD Biosciences) using the antibodies CD3 Pacific blue (UCHT1), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), and RORC2 Alexa Fluor 647 (Q31-378).
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