The following antibodies were used in the cross-presentation experiments: TruStain Fcblock anti-mouse CD16/32 (101320; BioLegend), FITC-CD11c (117306; Biolegend), APC-H2Kb-bound SIINFEKL (141606; Biolegend), APC/Cy7-CD40 (124637; Biolegend), PerCP/Cy5.5- ICAM_1 (116123; Biolegend), BV510-CD86 (563077; BD), PE/Cy7-MHC-II (107629; Biolegend), V450-CD80 (12519; BD). The following antibodies were used for the immunological analysis in the in vivo animal experiments: FITC-CD8 (553062; BD), PE-CD4 (100408; Biolegend), APC-CXCR3 (562266; BD), PerCP/Cy5.5-CD3 (100732; Biolegend), PE-CXCR3 (155903); FITC-CD8 (11083782); PerCP/Cy5.5-CD3 (100328);. Flow cytometric analyses were performed using Fortessa LSR Flow Cytometer (BD Biosciences) or BD Accuri C6 Plus (BD Bioscience). FlowJo software v.10 (FlowJo) was used for the data analysis.
Percp cy5.5 cd3
Manufactured by BD
Sourced in United States
PerCP-Cy5.5-CD3 is a fluorescent conjugate used in flow cytometry applications. It consists of the PerCP-Cy5.5 fluorochrome coupled to an antibody targeting the CD3 antigen, which is expressed on T cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 fitc, by BD (2 mentions)
Cd8 pe, by BD (2 mentions)
Cd4 fitc, by BD (2 mentions)
Cxcr3 pe, by BioLegend (1 mentions)
Pe cy 7 mhc 2, by BioLegend (1 mentions)
Cd11c fitc, by BioLegend (1 mentions)
Cd86 bv510, by BD (1 mentions)
Cd4 pe, by BioLegend (1 mentions)
Percp cy5.5 cd3, by BioLegend (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Il 17 pe, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Ifn γ apc, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Cd44 apc, by BD (1 mentions)
Lsr fortessa, by Tree Star (1 mentions)
Cd11c apc, by BD (1 mentions)
Cd38 bv711, by BD (1 mentions)
7 protocols using percp cy5.5 cd3
1
Cross-presentation Experiments with Antibodies
2
Cytokine Analysis of Vaccinated Splenocytes
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Flow cytometry was performed on spleen samples after vaccination on day 42. Lymphocytes were obtained from the cell suspension of the spleen by lymphocyte separation medium (n = 3 per group). Spleen mononuclear cells (1×106/mL) were cultured at 37°C and 5% CO2 with OS-TT+PT. Stimulation with PMA (250 ng/ml; Sigma-Aldrich) or medium only was used as positive and negative controls, respectively. The mononuclear cells (1×106 cells) were incubated with cell surface antibodies (PerCP-Cy5.5-CD3, FITC-CD4, PE-CD8, BD, USA) at 4°C for 30 min in the dark and then washed twice with PBS solution. To detect cytokines in splenocytes, the cells were incubated with FITC-CD4 in FACS buffer (1% BSA and 0.01% sodium azide) at 4 °C for 30 min. The cells were then washed 3 times with PBS. The cells were incubated with cell antibodies (PerCP-Cy5.5-IL-4, APC-IFN-γ, PE-IL-17, BD, USA) at 4°C for 30 min in the dark and then washed twice with PBS solution. Subsequently, the cells were analysed with a BD FACSCalibur™ Flow Cytometer.
3
Influenza NP-specific CD8+ T cell analysis
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MHC‐I tetramer targeting the immunodominant epitope of the influenza nucleoprotein (DbNP366–374 – ASNENMETM, DbPA224–233 – SSLENFRAYV) was produced in‐house and conjugated to streptavidin‐APC/PE (Life Technologies Australia Pty Ltd) at a 1:250 dilution at room temperature for 1h. Cells were stained with combinations of fluorochrome‐conjugated antibodies: PerCP‐Cy5.5‐CD3 (#551163), PE‐CD8 (#561095), BV421‐I‐Ab (#562928), APC‐CD44 (#553133), FITC‐CD44 (#553133), BV711‐CD38 (#740697), PerCP‐Cy5.5‐CD8 (#551162), APC/eF780‐CD62L (#47‐0621‐82), APC‐CD11c (#17011481), PE‐Cy7‐TCR‐va2 (#560624) from BD Biosciences, USA; and PE‐Cy7‐CD38 (#102718), BV785‐PD‐1 (#329908), APC‐Cy7‐CD45.1 (#110716), FITC‐I‐Ab (#116406), Pacific Blue‐I‐Ab (#116422), FITC‐CD19 (#115506) from Biolegend, USA. AF700‐CD3 (#56003382) was purchased from Invitrogen, USA.
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
4
NK Cell Cytotoxicity Assay
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K562 cells were propagated in RPMI/10 % FCS and sub-cultured 24 h before use. Cryopreserved PBMC were washed in RPMI and cultured in RPMI/5 % FCS at 1 × 106 cells/mL with K562 cells at a target:effector ratio of 10:1. After 6 h (37 °C), cells were transferred to flow tubes in PBS/1 %BSA, washed and 10 μL FcR Block (Miltenyi Biotech) was added for 20 min (4 °C). Cells were stained for extracellular markers [PerCP-Cy5.5-CD3, V450-CD56, V500-CD8, APC-CD107a clone H4A3] (BD Biosciences) for 15 min (room temperature). They were then washed, 250 μL Cytofix/Cytoperm (BD Biosciences) was added for 20 min (4 °C), followed by antibodies reactive with intracellular markers [FITC-perforin clone γG9; PE-IFNγ clone 4S.B3] (BD Biosciences) for 30 min. Washed cells were resuspended in PBS/1 % BSA and 500,000 events were acquired and analyzed (Additional file 1 : Figure S1).
5
Characterization of MICL Expression in Clec12A Knockout Mice
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MICL knockout mice (Clec12A−/−) on a C57BL/6 background were produced by Taconic Artemis, USA (see online supplementary figure S1A). Clec12A−/− and wild-type (wt) mice were bred and maintained at the Medical Research Facility, University of Aberdeen. Mice were separately housed in groups and provided freely with food and water. All experimentation conformed to the terms and conditions of UK Home Office licences for research on animals (PPL 60/4007) and the University of Aberdeen ethical review committee.
Characterisation of MICL expression in 8–12-week-old wt and Clec12A−/− mice was performed by flow cytometry on cells isolated from the peripheral blood, bone marrow, peritoneal cavity, spleen and lungs, as previously described.14 (link) Antibodies used in these experiments included biotin anti-MICL monoclonal antibody 3097 (link) and isotype control, as well as biotin-Gr-1, FITC-7/4, PE-F480, biotin-F480, PerCpCy5.5-CD11b, PE-CCR3, biotin-NK1.1, PE-CD49, PerCpCy5.5-B220, PE-CD19, PerCpCy5.5-CD3, biotin-CD4, FITC-CD8, biotin-CD11c, PerCpCy5.5-Gr-1, APC-Ly6G, PE-CD11b, PE-Ly6G, PECy7-CD11b, Alexa Fluor 488 anti-STAT5, APC-streptavidin (all from BD Biosciences), and Alexa Fluor 700-F4/80 (BioLegend). Flow cytometry was undertaken using a BD LSRFortessa cell analyser and data analysed using FlowJo.
Characterisation of MICL expression in 8–12-week-old wt and Clec12A−/− mice was performed by flow cytometry on cells isolated from the peripheral blood, bone marrow, peritoneal cavity, spleen and lungs, as previously described.14 (link) Antibodies used in these experiments included biotin anti-MICL monoclonal antibody 3097 (link) and isotype control, as well as biotin-Gr-1, FITC-7/4, PE-F480, biotin-F480, PerCpCy5.5-CD11b, PE-CCR3, biotin-NK1.1, PE-CD49, PerCpCy5.5-B220, PE-CD19, PerCpCy5.5-CD3, biotin-CD4, FITC-CD8, biotin-CD11c, PerCpCy5.5-Gr-1, APC-Ly6G, PE-CD11b, PE-Ly6G, PECy7-CD11b, Alexa Fluor 488 anti-STAT5, APC-streptavidin (all from BD Biosciences), and Alexa Fluor 700-F4/80 (BioLegend). Flow cytometry was undertaken using a BD LSRFortessa cell analyser and data analysed using FlowJo.
6
Isolation and Phenotyping of Lung Immune Cells
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Mice were sacrificed on day 3 or 5 post-challenge and immediately perfused with sterile PBS through the right ventricle to eliminate the interference of cells in pulmonary vessels. Next, the lungs were excised, smashed, and enzymatically digested using the mouse lung dissociation kits (Miltenyi Biotec, Germany). Lung cells were blocked with CD16/32 antibody (BioLegend, Cat. No.156604) and stained on ice in the dark with surface antibodies, including PerCP-Cy5.5-CD3 (BD Bioscience, Cat. No. 560527), FITC-CD4 (BD Bioscience, Cat. No. 553650), APC-CD8 (BioLegend, Cat. No. 100712), and PE-CD69 (BioLegend, Cat. No. 104508). After staining, cells were acquired on a CytoFLEX flow cytometer (Beckman coulter) and analyzed using FlowJo software. The gating strategy is shown in Supplementary Figure 3
7
Multicolor Flow Cytometry of Spleen Macrophages
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1 to 2 × 106 freshly thawed spleen cells were surface stained using a cocktail of fluorescent-labelled antibodies – PerCP/Cy5.5-CD3 (Clone SP34-2, BD Biosciences), BV510-CD20 (Clone 2H7, Biolegend), APC/Cy7-HLA-DR (Clone L243, Biolegend), AF700-CD14 (Clone M5E2, Biolegend), APC-TLR4 (Clone HTA125, Biolegend), PB-CD16 (Clone 3G8, Biolegend), PE-CX3CR1 (Clone 2A9-1, Biolegend), PerCP-CCR2 (Clone 48,607, R&D Systems), BV605-CD40 (Clone 5C3, Biolegend), and PE-Dazzle 594-CD80 (Clone 2D10, Biolegend). All samples were acquired with the Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA) and analysed using FlowJo software (Ashland OR). Median Fluorescence Intensities (MFI) for all markers within the CD14+ macrophage gate were extracted in FlowJo and tested for significant differences using an unpaired t-test with Welch's correction on Prism 5 (GraphPad, San Diego CA).
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