Expression of IL-2, IFNγ and IL-17 by mononuclear cells in response to restimulation by Ag85A and ESAT-6 peptides was assayed by ICS, and data were analyzed using a hierarchical gating strategy, as outlined elsewhere [17 (link), 19 (link)]. Fluorochrome antibodies used for staining included CD3-APC-eFluor780 (Invitrogen), CD4-APC, CD8-PE-Cy7, IFNγ-PerCP Cy5.5, IL-17A-PE/Dazzle 594 (all from Biolegend) and IL-2-PE (BD Pharmingen). For tetramer staining, single cell suspensions of 1–2 × 106 cells per well were stained with the fluorescent antibodies CD3e-Pacific Blue and CD8-FITC (BD Pharmingen) and the PE-labeled MVPGGQSSF/H-2d tetramer (MHC Tetramer Production Facility, Baylor College of Medicine, Houston, Texas) for 45 minutes at room temperature. Cells were then washed twice with PBS, fixed, and 200,000 events were acquired on the FACS Canto II (Beckman Coulter).
Cd3 apc efluor780
Manufactured by Thermo Fisher Scientific
Sourced in Czechia, United States
The CD3-APC-eFluor780 is a fluorescently-labeled antibody that binds to the CD3 antigen on the surface of T cells. It can be used for the identification and enumeration of T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd19 apc efluor780, by Thermo Fisher Scientific (3 mentions)
Lsrfortessa, by BD (3 mentions)
Cd16 pe cy7, by BioLegend (2 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus media, by Thermo Fisher Scientific (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Cd4 efluor450, by Thermo Fisher Scientific (2 mentions)
Cd137 pe cyanine7, by BioLegend (2 mentions)
Cd57 bv605, by BioLegend (2 mentions)
Cd7 bv786, by BioLegend (2 mentions)
Cd16 buv496, by BD (2 mentions)
Cd337 nkp30 bv421, by BD (2 mentions)
Cd159a nkg2a biotin pe vio770, by Miltenyi Biotec (2 mentions)
Cd159c nkg2c pe, by Miltenyi Biotec (2 mentions)
Cd337 nkp30 efluor 450, by Thermo Fisher Scientific (2 mentions)
Cd14 apc efluor 780, by Thermo Fisher Scientific (2 mentions)
Prism v8, by GraphPad (2 mentions)
Cd56 pe dazzle 594, by BioLegend (2 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
14 protocols using cd3 apc efluor780
1
Cytokine Profiling of Ag85A and ESAT-6 Responses
2
Flow Cytometry Antibody Panel for Immune Profiling
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The following antibodies were used for flow cytometry: purchase from Invitrogen: CD3-APC-efluor780 (145-2C11), CD4-AlexaFluor 700 (GK1.5), CD4-Pacific Orange (RM4-5), CD8-Super Bright 645 (53-6.7), NKp46-eFluor 660 or AlexaFluor 647(29A1.4), NK1.1-Super Bright 436 (PK136), GATA-3-PeCy5 (TWAJ), PD-1-SuperBright 780 (J43), Foxp3-AlexaFluor 488 (FJK-16s), CD103-AlexaFluor700 (2E7), RoRγt-PE-eFluor610 (B2D), PLZF-PeCy7 (9E12), TNF-α-PerCP-eFluor710 (MP6-XT22), IL-17-PeCy7 (eBio17B7), IL-13-Pe-eFluor610 (eBio13A), CD127-PeCy5 (A7R34), Granzyme B-eFluor450 (NGZB); purchased from Biolegend: T-bet-Brilliant Violet 605 (4B10), CD69-Brilliant Violet 711 (H1.2F3), CD44-Brilliant Violet 421 (IM7), IFN-γ-Brilliant Violet 510 (XMG1.2), TCR γδ-Pe (UC7-13D5), IL-4-Brilliant Violet 711 (11B11), IL-22-APC (Poly5164), ki67-PerCP/Cy5.5 (16A8), CXCR3-Brilliant Violet 510 (CXCR3-173), CXCR6-Brilliant Violet 421 (SA051D1), Perforin-Pe (S16009B), B220-Brilliant Violet 750 (RA3-6B2), F4/80-Brilliant Violet 785 (BM8), Ly6G/Ly6C-Alexa Fluor 700 (RB6-8C5). Appropriate isotype controls were also purchased.
The α-GalCer-CD1d-tetramer-AlexaFluor 488, the MHC class I NS41602-1611 tetramer-APC and the MHC class I NS31043-1052 tetramer PE were obtained from the NIH tetramer core facility.
The α-GalCer-CD1d-tetramer-AlexaFluor 488, the MHC class I NS41602-1611 tetramer-APC and the MHC class I NS31043-1052 tetramer PE were obtained from the NIH tetramer core facility.
3
Multiparametric Flow Cytometry Analysis
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BALF cells and PBMCs were stained for extra- and intracellular markers using the following antibodies: CD3-APC-eFluor780(SK7), CD4-AF700(OKT4), CD45RO-FITC(UCHl-1), CD95-APC(DX2), FoxP3-APC(PCH101) (eBiosciences) and CD25-PE(M-A251), CD25-PE-Cy7(M-A251), CD127-V450(hIL7R-M21) anti-CTLA-4-BV421(BNI3) (BD biosciences) and CD45RA-PE-Texas Red(MEM-56) (Invitrogen). Fixable Aqua Dead Cell Stain kit for 405 nm (Invitrogen, Molecular Probes) was used as live-dead marker. Cells were measured on a Flow cytometer LSRII (BD Biosciences).
4
Differential Immunophenotyping of NK Cell Subsets
5
Isolation and Analysis of Skin Lymphocytes and DCs
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Isolation and analysis of skin lymphocytes [18 (link), 27 (link)] were carried out as described. Briefly, after removing fur with an electric groomer, trunk skin was separated from subcutaneous fat tissue, cut to small pieces and, for lymphocyte analysis, digested for 45 min. at 37°C in RPMI 1640 containing collagenase XI (4000 U/ml), hyaluronidase (260 U/ml) and DNase (0.1mg/ml), all from Sigma-Aldrich. Cells were harvested by filtration, washed and stained, first for surface markers with mAbs CD45.2-PerCP, CD3-APC-eFluor780, CD4-FITC, CD25-PE, CTLA4-APC, ICOS-PE-Cy7, and live/dead-eFluor506, then fixed and permeabilized and stained with FoxP3-eFluor450, all from eBioscience. For DC analysis, digestion was with 150μg/ml Liberase and 120μg/ml in HBSS and cells were stained with mAbs CD11c-PerCPCy5.5 and CD103-PE (both BioLegend) and, after fixation and permeabilization, with anti-langerin-Alexa488 (clone 929F3.01, Dendritics).
6
Isolation and Analysis of Murine Immune Cells
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Spleen, MLN and kidney were collected and mashed in 70-μm cell strainers with C10 media (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, all from Life Technologies, Grand Island, NY). For splenocytes, red blood cells were lysed with RBC lysis buffer (eBioscience, San Diego, CA). To isolate intestinal epithelial cells (IECs), the entire intestine including small intestine and colon was opened longitudinally and cut into pieces. The pieces were incubated twice in EDTA-DTT solution and intensively vortexed to harvest IEC-enriched fractions. For surface marker staining, cells were blocked with anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with Attune NxT flow cytometer (Thermo Scientific). For intracellular staining, Foxp3 Fixation/Permeabilization kit (eBioscience) was used. Anti-mouse antibodies used in this study include: CD3-APC-eFluor 780, CD8-PE-Cy7, CD4-PerCP-Cy5.5, RORγT-PE, CD3e-biotin (eBioscience); CD45-APC-Cy7, IL-10-BV421, IL-17A-APC, CD49b-biotin, CD19-biotin (Biolegend, San Diego, CA); Biotin-FITC (MACS). Flow cytometry data were analyzed with FlowJo.
7
Characterization of NK Cell Receptors
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PBMC were obtained from buffy coats of healthy donors (n=4) by Ficoll-Paque™ PLUS Media gradient centrifugation (Thermo Fisher Scientific, USA). PBMC were treated as described above and the expression of activating and inhibitory receptors on NK cells was determined by flow cytometry on day 3 and 7 using following fluorescently labeled antibodies in two panels: CD45-PE-Dy594 (Exbio, Czech Republic), CD3-APC-eFluor780 (eBioscience, USA), NKG2D-PE-Cy7, CD56-Alexa Fluor700, CD16-PE-Cy7, DNAM-1-FITC (all from Biolegend, USA), NKp30-PE, NKG2A-APC (R&D), CD158a-PE and CD158b-FITC (BD Biosciences, USA). The dead cells detected by LIVE/DEAD Fixable Aqua stain (Thermo Fisher Scientific, USA) were excluded from the analyses. The gating strategy was as follows: singlets/live CD45+/CD3-/CD56+/receptor+. Mean fluorescence intensity (MFI) of receptor expression on NK cells was determined from CD3-CD56+ cell population.
8
Multiparametric Flow Cytometry Analysis
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The following antibodies were purchased from BD: Ki67 AF700, CD4 Pacific Blue, CD8α PE-CF594, CD3 PE-CF594, CD4 BV650, and CD11a BUV805. The following antibodies were purchased from eBioscience: KLRG-1 FITC, Ly6C (clone HK1.4) PerCP-Cy5.5, CD69 eFluor450, CD69 FITC, CD69 PE-Cy7, T-bet eFluor660, CD45.2 APC-eFluor780, and CD3 APC-eFluor780. The following antibodies were purchased from BioLegend: LFA-1 PerCP-Cy5.5, CD3 Pacific Blue, CD3 BV785, CD11a PE, and CD11a PErCP-Cy5.5. Invitrogen live/dead Aqua stain or Tonbo Ghostdye510 was used to determine viability. PE-conjugated Tgd-057 MHC-I tetramers and PE-conjugated AS-15 MHC-II tetramers were provided by NIH Tetramer Core. All samples were run on an LSRFortessa (BD), Canto (BD), or FACSymphony A3 and analyzed using FlowJo software (Tree Star). Images were obtained using the ImageStream (Amnis) and analyzed using IDEAS software (Amnis). To determine T-bet localization, nuclear and cytoplasmic masking functions were made using DAPI staining; these masks were then applied to T-bet expression.
9
Multicolor Flow Cytometry Panel
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The following fluorochrome conjugated anti-human antibodies were used: CD3 APC efluor 780 (eBioscience #47-0036-42) or BV650 (Biolegend #317324), CD4 BV 510 (BD Horizon/BD Biosciences #562970), CD8 evolve 655 (eBioscience #86-0088-42), CD33 APC (eBioscience#17-0338-42), CD56 efluor 710 (eBioscience #46-056-42) or FITC (Biolegend #304604), PD-1 BV 785 (BioLegend #329930), CCR7 BV785 (Biolegend #353230), CD19 BV785 (Biolegend #302240), CD62L BV785 (Biolegend #304830), IFN-γ efluor 450 (eBioscience #48-7319-42), Perforin Alexa Fluor 647 (Biolegend #353322) or PE (Biolegend #353304), granzyme FITC (Biolegend #515403). Appropriate isotype control antibodies were employed to determine the specificity of test antibodies.
10
Expansion of NK Cells by SOT101
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Peripheral blood mononuclear cells (PBMC) were obtained from buffy coats of healthy donors (n=8) by Ficoll-Paque™ PLUS Media gradient centrifugation (Thermo Fisher Scientific, USA). PBMC were cultivated in RPMI 1640 medium supplemented with 2 mM GlutaMAX I CTS, 100 U/ml penicillin + 100 mg/ml streptomycin, 1 mM Sodium pyruvate, 1% non-essential amino acids, 50 μM 2-Mercaptoethanol (all from Gibco, Thermo Fisher Scientific, USA) and 10% AB human serum (heat-inactivated) (Invitrogen, USA). 1 × 106/ml PBMC were incubated with SOT101 at concentrations of 0, 0.001, 0.01, 0.1, 1 and 10 nM for 7 days at 37°C in a humidified atmosphere containing 5% CO2. The phenotype of NK cell populations and their relative expansion and proliferation was determined by flow cytometry on day 3 and 7 using following fluorescently labeled antibodies: CD3-APC-eFluor780, CD4-eFluor450, Ki67-APC (all three from eBioscience, USA), CD8-PE-DlyLight594 (Exbio, Czech Republic), CD56-Aexa Fluor700, CD16-PE-Cy7 (both from Biolegend, USA). The dead cells detected by LIVE/DEAD Fixable Aqua stain (Thermo Fisher Scientific, USA) were excluded from the analyses. The gating strategy was as follows: singlets/live lymphocytes/CD3-/CD56 vs. CD16 or CD56+Ki67+.
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