The following antibodies were used for staining: CD4 PerCp-Cy5.5, TCRβ PerCp-Cy5.5, and CD62L APC (TONBO); CD44 FITC, PD-1 (29F.1A12) PE-Cy7, CD69 PE-Cy7, CD5 PerCp-Cy5.5, CD8α Pacific Blue, and TCRβ Pacific Blue (BioLegend); CD44 PE-Cy7, CD8α (APC-eFluor 780), CD4 Qdot606, and Zap70 FITC (Life Technology). In vivo anti-PD (J43) antibody and control IgG were purchased from BioXcell. For intracellular flow cytometry, antibodies against phosphorylated ERKT202/Y204 (197G2), AKTThr308 (C31E5E), and ribosomal protein S6Ser235/236 (D57.2.2E) were purchased from Cell Signaling Technology.
Cd69 pe cy7
Manufactured by BioLegend
Sourced in United States
CD69-PE/Cy7 is a fluorescently-labeled antibody that binds to the CD69 cell surface marker. CD69 is an early activation antigen expressed on activated T cells, B cells, and NK cells. The PE/Cy7 fluorescent dye combination provides a bright signal and good separation from other fluorescent dyes commonly used in flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Facs symphony a5, by BD (2 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Ly6g apc, by BioLegend (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Cd11b percp cy5, by BioLegend (2 mentions)
Pma ionomycin, by BioLegend (2 mentions)
B220 bv605, by BioLegend (2 mentions)
Live dead apc cy7, by BioLegend (2 mentions)
Cd94 bv421, by BioLegend (2 mentions)
Cd4 pe, by Thermo Fisher Scientific (2 mentions)
B220 apc, by BioLegend (2 mentions)
Golgiplug, by BD (2 mentions)
Cd3 fitc, by BioLegend (2 mentions)
F4 80 pe, by BioLegend (2 mentions)
Cd3 apc cy7, by BioLegend (2 mentions)
Cd44 apc, by BioLegend (2 mentions)
Cd45 bv421, by BioLegend (2 mentions)
Cd4 pe, by BioLegend (2 mentions)
Cd11b bv510, by BioLegend (2 mentions)
Gr1 apc cy7, by BioLegend (2 mentions)
20 protocols using cd69 pe cy7
1
Multi-Marker Immune Cell Analysis
2
Hamster PBMC Isolation and Activation
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Hamster peripheral blood mononuclear cells (PBMCs) were isolated from ethylene diamine tetraceticacid (EDTA) whole blood by overlay on a Histopaque®-1077 density cushion and separated according to manufacturer’s instructions. Tissues were processed into single cell suspensions as described previously (44 (link)). Cells were stimulated for 6 hours with media alone, cell stimulation cocktail (containing PMA-Ionomycin, Biolegend), 1μg/ml SARS-CoV-2 S peptide pool (IDT), or Lassa virus (LASV) GPC peptide pool (IDT) together with 5μg/ml Brefeldin A (Biolegend). Following surface staining with Live/Dead-APC/Cy7, CD4-FITC, CD8-Alexa700, CD94-BV421 and CD69-PeCy7, B220-BV605, CD11b-PerCPCy5.5, and Ly6G-APC (all Biolegend) cells were fixed with 4% paraformaldehyde (PFA). Sample acquisition was performed on a FACSSymphony-A5 (BD), and data analyzed in FlowJo V10. Cell populations were identified by initially gating on Live/Dead negative, doublet negative (SSC-H vs SSC-A). Activation positive responses are presented after subtraction of the background responses detected in the LASV GPC peptide pool-stimulated samples.
3
Multicolor Flow Cytometry Panel
4
Multi-Parameter Flow Cytometry of T Cells
5
Comprehensive Immune Cell Profiling
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The cells were extracted from lymph nodes, spleen, or colon tissue digested by collagenase. Isolated cells were stained with following antibodies: CD8-PE-CY7 (Thermo Scientific), CD4-APC-Fire™ 750 (BioLegend), IFN-γ-FITC (BioLegend), IL-17-APC (BioLegend), FOXP3-PE (BioLegend), CD19-PE (BioLegend), NK1.1-FITC (BioLegend), CD11B-APC (BioLegend), Gr-1-FITC (BioLegend), F4/80-PE (BioLegend), CD11C-APC-CY7 (BioLegend), MHC-II-PE/Dazzle™ 594 (BioLegend), CD8-APC (BioLegend), Ki67-PerCP-CY5.5 (BioLegend), Annexin V-FITC (BioLegend), CD103-PE (BioLegend), CD69-PE-CY7 (BioLegend), CD44-PE-CY7 (BioLegend), CD62L-FITC (BioLegend), CD107-FITC (BioLegend), IFN-γ-APC (BioLegend), CD107-PE (BioLegend), IFN-γ-PE (BioLegend), CD3-FITC (BioLegend), CD3-APC/Fire™ 750 (BioLegend), CD8-APC (BioLegend), IFN-γ-PE-CY7 (BioLegend), CD69-APC/Fire™ 750 (BioLegend), Tetramer-SIINFEKL-PE (MBL), p-JNK-PE (Cell Signaling Technology), p-IRAK4-Alexa Fluor 488 (Cell Signaling Technology). Intracellular staining was carried out using the Cytofix/Cytoperm kit (BD Pharmingen) following a 4 h restimulation by PMA/ionomycin (Sigma) in the presence of GolgiPlug (BD Pharmingen). Flow cytometric data acquisition was performed on a NovoExpress flow cytometer and analyzed with NovoExpress software (ACEA Biosciences).
6
Phenotypic Analysis of Influenza-Specific T Cells
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Mononuclear cells were washed twice with cold PBS with 0.5% FBS and 0.1% NaN3 and centrifuged at 1,600 rpm for 5 min at 4°C. Purified rat anti-mouse CD16/CD32 (BD Pharmingen, San Diego, CA, USA) Fc blocker and 5 µg/ml streptavidin (Invitrogen) were added to each sample and incubated for 20 min at 4°C. The following cocktail was used for surface staining: CD3-APC/Cy7 (Clone 17A2; BioLegend), CD8-PE (Clone KT15; MBL Life science, Tokyo, Japan), CD62L-Alexa Fluor 700 (Clone MEL-14; BioLegend), CD69-PE/Cy7 (Clone H1.2F3; BioLegend), CD103-FITC (Clone 2E7; BioLegend) and influenza B-NP specific Dd/NP166-174 (FSPIRITFL) tetramer. The B-NP-specific Dd/NP166-174 (FSPIRITFL) tetramer was produced as described previously (20 (link)). In brief, recombinant Dd protein was refolded in the presence of NP166-174 peptide and β2m, biotinylated with biotin-protein ligase BirA, and then purified with Superdex-75 gel filtration column (GE Healthcare Life science, Chicago, IL, USA). The purified monomer was tetramerized with Alexa Fluor 647-streptavidin conjugates (Thermo Fisher Scientific, Waltham, MA, USA). Stained cells were washed, fixed using BD FACS Lysing Solution (BD Pharmingen) at RT for 20 min, and collected on an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed using Flowjo software (TreeStar Inc., Ashland, OR, USA).
7
Flow Cytometric Analysis of T-Cell Activation
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Harvested cells were stained for flow cytometry in PBS for 20 min at RT. The following antibodies were purchased from BioLegend (San Diego, United States) and used for flow cytometric analysis: CD8a-BV510 (cat: 100752, clone: 53-6.7, dilution 1:200), CD45.1-PacificBlue (cat: 110722, clone: A20, dilution 1:200), CD44-APC (cat: 559250, clone: IM7, dilution 1:100), CD11c-PerCP (cat: 117326, clone: N418, dilution 1:200), CD69-PeCy7 (cat: 104512, clone: H1.2F3, dilution 1:100), CD25-FITC (cat: 553072, clone: 3C7, dilution 1:100). The viability of cells was determined by using fixable near-IR dead cell staining (LifeTechnologies, Carlsbad, United States). Biotinylation was assessed by streptavidin-PE (BioLegend) staining for 10 min at RT. T-cell activation was determined by the expression of GFP expression under the control of the Nur77 promoter. Data was acquired on a CantoTM flow cytometer (BD Bioscience, Allschwil, Switzerland) and analyzed using the FlowJo software (Treestar, Ashland, OR, USA) (see Supplementary Fig. 12 for gating strategy).
8
Hamster PBMC Isolation and Immune Response
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Hamster PBMCs were isolated from ethylene diamine tetraceticacid (EDTA) whole blood by overlay on a Histopaque®−1077 density cushion and separated according to manufacturer’s instructions. Tissues were processed into single cell suspensions as described previously (Barrigan et al., 2013 (link)). Cells were stimulated for 6 hours with media alone, cell stimulation cocktail (containing PMA-Ionomycin, Biolegend), 1μg/ml SARS-CoV-2 S peptide pool, or Lassa virus (LASV) GPC peptide pool together with 5μg/ml Brefeldin A (Biolegend). Following surface staining with Live/Dead-APC/Cy7, CD4-Alexa700, CD8-FITC, CD94-BV421 and CD69-PeCy7, B220-BV605, CD11b-PerCPCy5.5, and Ly6G-APC (all Biolegend) cells were fixed with 4% paraformaldehyde (PFA). Sample acquisition was performed on a FACSSymphony-A5 (BD), and data analyzed in FlowJo V10. Cell populations were identified by initially gating on Live/Dead negative, doublet negative (SSC-H vs SSC-A). Activation positive responses are presented after subtraction of the background responses detected in the LASV GPC peptide pool-stimulated samples.
9
Multicolor Flow Cytometry Protocol
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Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45‐vioblue 450, CD8‐FITC, CD19‐APC (Tonbo, San Diego, CA), HLA‐DR‐FITC, CD3‐viogreen, CD103‐PE (Miltenyi Biotec, Auburn, CA, USA), CD11c‐PerCp‐Cy5.5, CD69‐PE‐Cy7 (Biolegend, San Diego, CA), CD103‐PE‐Cy7, CD4‐PE (eBiosciences, San Diego, CA, USA), CD56‐APC (BD Pharmingen, San Diego, CA, USA). Dead cells were excluded with 7AAD (Southern Biotech) or zombie dye yellow staining (Biolegend). Analysis was performed on 8‐color MACSQuant 10 (Miltenyi biotech) or Gallios (Beckman Coulter) flow cytometers and data were analyzed with FlowJo software (Tree Star, Inc. Ashland, OR, USA). Expression of surface markers is shown as percentage of positive cells and MFI. Fluorescence minus one (FMO) strategy was used to establish appropriate gates. Comparisons between FMO and isotype controls showed no differences. For tissue dendritic cell (DC) quantification, DCs were identified using flow cytometry as CD45+, CD3‐, CD19‐, CD56‐, HLA‐DRhigh, CD11c+ cells as described before (Rodriguez‐Garcia et al., 2017 ), and the cell number was normalized to tissue weight.
10
Multiparameter Flow Cytometry Characterization
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The characterization of differentiated MDM was performed via flow cytometry with the following mAbs: CD11b BV 786 (Biolegend, San Diego, CA, USA; clone IRCF44), CD14 APC (BD Biosciences, Franklin Lakes, NJ, USA; clone M5E2), CD206 FITC (BD Biosciences, clone 19.2), HLA-DR BV711 APC (BD Biosciences, clone G46-6), CD16 PE (BD Biosciences, clone 3G8), CD4 BV 421 (BD Biosciences, clone RPA-T4), CD8FITC (BD Biosciences, clone RPA-T8), CD19 APC (BD Biosciences, cloneHIB19) and CD69PECy7 (Biolegend, clone G10F5). Live dead staining (ThermoFisher, Waltham, MA, USA) was performed as a control for vitality. Labelled cells were fixed in PBS−/− 1× 1% formalin. A minimum of 50.000 events were acquired for each sample using a BD Lyric (BD Biosciences) and analyzed by FACS Diva 8.0.1 (BD Biosciences).
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