Cultured T cells were washed in PBS/0.1% BSA and re-suspended at 1×106 cells in 50 uL Brilliant Buffer (BD Biosciences) supplemented with 4% rat serum for 15 minutes at 4C. Cells were then incubated for 30 minutes at 4°C in 100 μL of Brilliant Buffer using the following antibody fluorophore conjugates (all from BD Biosciences unless otherwise noted): CD7 BV421, CD4 BV510, CCR4 BV605 (BioLegend), CD8 BV650, CD196 BV786 (BioLegend), CD3 AF488, CD45RA PerCPCy5.5, CD183 PE, CD197 PE-CF594, CD185 PE-Cy7 (BioLegend), and CCR10 APC (R&D Systems). Full details of fluorophore conjugated antibodies can be found in supplemental table 5 . Cells were then washed twice in PBS/0.1% BSA and data acquired on a ZE5 (Yeti) cytometer (BioRad/Propel Labs). Compensation and analyses were performed on FlowJo V10 (TreeStar) using fluorescence minus one (FMO) controls. Statistical analyses were performed on GraphPad Prism 7 using 2-way ANOVA with Bonferroni post-hoc corrections.
Cd8 bv650
Manufactured by BioLegend
The CD8 BV650 is a fluorescently-labeled monoclonal antibody that binds to the CD8 antigen on the surface of T cells. It is used in flow cytometry applications to identify and quantify CD8-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 bv605, by BioLegend (4 mentions)
Live dead aqua, by Thermo Fisher Scientific (3 mentions)
Ccr10 apc, by R&D Systems (2 mentions)
Cd4 bv510, by BioLegend (2 mentions)
Ccr4 bv605, by BioLegend (2 mentions)
Cd3 af488, by BioLegend (2 mentions)
Cd45ra percp cy5, by BioLegend (2 mentions)
Cd183 pe, by BioLegend (2 mentions)
Brilliant buffer, by BD (2 mentions)
Cd7 bv421, by BioLegend (2 mentions)
Cd196 bv786, by BioLegend (2 mentions)
Cd197 pe cf594, by BioLegend (2 mentions)
Cd185 pe cy7, by BioLegend (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Cd3 percp cy5, by BioLegend (2 mentions)
Il 17a pe, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
13 protocols using cd8 bv650
1
Multiparameter Flow Cytometry of T Cells
2
Comprehensive Immunophenotyping of T Cells
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Cryopreserved peripheral blood mononuclear cells were thawed and stained in Horizon Brilliant Stain Buffer (BD) containing all antibodies and Live/Dead Near-IR (Life Technologies) at 1:300 dilution and stained at 4°C for 30 minutes in Horizon. Panel 1 included the following: CD3 BV570 (UCHT1), CCR7 Pacific Blue (G043H7), and CD27 AlexaFluor700 (M-T271) (all BioLegend); CD4 BV605 (RPA-T4) and CD8 BV650 (RPA-T8) (BD); programmed cell death protein 1 (PD-1) phycoerythrin (PE)–eFluor610 (eBioJ105), CD45RA fluorescein isothiocyanate (HI100), and T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) peridinin-chlorophyll protein complex (PerCP)–eFluor710 (MBSA43) (eBioscience); and Tim-3 PE (344823) (R&D).
Panel 2 included the following: CD3 BV570, CD38 AlexaFluor700 (HB-7) (BioLegend); CD4 BV605, CD8 BV650, PD-1 PE-eFluor610, and Tim-3 PE. After this, cells were washed twice before fixation and permeabilization with Foxp3 Buffer Set (BD). Staining for intracellular epitopes was performed with: T-bet fluorescein isothiocyanate (4B10) (BioLegend) and Eomes eFluor660 (WD1928) (eBioscience). Samples were acquired on an LSR II flow cytometer (BD). Data were analyzed using FlowJo software (version 10.8.0r1; Tree Star).
Panel 2 included the following: CD3 BV570, CD38 AlexaFluor700 (HB-7) (BioLegend); CD4 BV605, CD8 BV650, PD-1 PE-eFluor610, and Tim-3 PE. After this, cells were washed twice before fixation and permeabilization with Foxp3 Buffer Set (BD). Staining for intracellular epitopes was performed with: T-bet fluorescein isothiocyanate (4B10) (BioLegend) and Eomes eFluor660 (WD1928) (eBioscience). Samples were acquired on an LSR II flow cytometer (BD). Data were analyzed using FlowJo software (version 10.8.0r1; Tree Star).
3
Multiparameter Flow Cytometry of T Cells
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Cultured T cells were washed in PBS/0.1% BSA and re-suspended at 1×106 cells in 50 uL Brilliant Buffer (BD Biosciences) supplemented with 4% rat serum for 15 minutes at 4C. Cells were then incubated for 30 minutes at 4°C in 100 μL of Brilliant Buffer using the following antibody fluorophore conjugates (all from BD Biosciences unless otherwise noted): CD7 BV421, CD4 BV510, CCR4 BV605 (BioLegend), CD8 BV650, CD196 BV786 (BioLegend), CD3 AF488, CD45RA PerCPCy5.5, CD183 PE, CD197 PE-CF594, CD185 PE-Cy7 (BioLegend), and CCR10 APC (R&D Systems). Full details of fluorophore conjugated antibodies can be found in supplemental table 5 . Cells were then washed twice in PBS/0.1% BSA and data acquired on a ZE5 (Yeti) cytometer (BioRad/Propel Labs). Compensation and analyses were performed on FlowJo V10 (TreeStar) using fluorescence minus one (FMO) controls. Statistical analyses were performed on GraphPad Prism 7 using 2-way ANOVA with Bonferroni post-hoc corrections.
4
Cytokine Analysis of Activated T Cells
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Cells used for in vitro or in vivo cytokine analysis were stimulated with PMA (20 ng/ml; Sigma-Aldrich), Ionomycin (1 µg/ml; Sigma-Aldrich), and Brefeldin A (2 µg/ml; Sigma-Aldrich) for 4 hr prior to flow staining (Lu et al., 2015 (link)). For intracellular staining, cells were fixed and permeabilized using the Mouse FOXP3 Buffer Set (BD) per the manufacturer’s protocol. The fluorophore-conjugated antibodies used in this study were as follows: Live/Dead Fix Blue (Invitrogen), CD3 PerCPCy5.5 (Biolegend), TCRb PE/Cy7 (Biolegend), CD4 PB (Biolegend), CD4 AF700 (Biolegend), CD8 BV650 (Biolegend), CD25 BV421 (Biolegend), FOXP3 AF488 (Biolegend), ROR gamma T PE (Invitrogen), TCF1 AF647 (Cell Signaling Technologies), TCF1 PE (Biolegend), IFNγ AF647 (Biolegend), IL17A FITC (Biolegend), and IL17A PE (ebioscience). Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).
5
Immunophenotyping of Expanded T Cells
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In vitro expanded cells were washed, counted, and plated in a 96 well plate at a density of 1 × 106 cells/well. Cells were then stained with a mixture of the following antibodies: Fixable Viability Dye eFluor 506 (Thermo Fisher), CD3-AF700 (BD, RRID:AB_10597906), CD4-BV711 (BD, RRID:AB_2740432), and CD8-BV650 (Biolegend, RRID:AB_11125174) for 30 min at 4°C in the dark. Stained cells were then washed twice and resuspended in 100 μL PBS to be run on an LSR II flow cytometer (BD; a detailed protocol can be found at (https://doi.org/10.17504/protocols.io.bwu9pez6 ). FCS files produced from the LSR-II were then analyzed using FlowJo v10.8.2 software (Tree Star; RRID:SCR_008520; https://www.flowjo.com/solutions/flowjo ).
6
SARS-CoV-2 Specific T Cell Activation Assay
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The AIM assay was performed as previously described25 (link),50 (link). Cryopreserved PBMCs were thawed by diluting the cells in 10 mL complete RPMI 1640 with 5% human AB serum (Gemini Bioproducts) in the presence of benzonase [20 μl/10ml]. Cells were cultured for 20 to 24 hours in the presence of SARS-CoV-2 specific MPs [1 μg/ml], mesopools [1 μg/ml], 15-mers [10 μg/ml], or class I predicted peptides [10 μg/ml] in 96-wells U bottom plates with 1x106 PBMC per well. As a negative control, an equimolar amount of DMSO was used to stimulate the cells as a negative control in triplicate wells, and phytohemagglutinin (PHA, Roche, 1 μg/ml) was included as the positive control. The cells were stained with CD3 AF700 (4:100; Life Technologies Cat# 56-0038-42), CD4 BV605 (4:100; BD Biosciences Cat# 562658), CD8 BV650 (2:100; Biolegend Cat# 301042), and Live/Dead Aqua (1:1000; eBioscience Cat# 65-0866-14). Activation was measured by the following markers: CD137 APC (4:100; Biolegend Cat# 309810), OX40 PE-Cy7 (2:100; Biolegend Cat#350012), and CD69 PE (10:100; BD Biosciences Cat# 555531). All samples were acquired on either a ZE5 cell analyzer (Bio-rad laboratories) or an Aurora flow cytometry system (Cytek), and analyzed with FlowJo software (Tree Star).
7
SARS-CoV-2 Spike Protein Peptide Stimulation
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Cryopreserved PBMC were thawed and rested for 4 hours in RPMI-1640 supplemented with 10% FCS and penicillin-streptomycin (RF10). 2x10 6 PBMC were seeded per well in a 96-well U bottom plate and stimulated with 1ug/mL of a peptide pool covering the spike protein (PepTivator SARS-CoV-2 Prot_S Complete) or an equivalent volume of vehicle control (sterile H2O). After 1 hour, Brefeldin A (Golgi Plug, BD Biosciences) was added to the cell culture. PBMC were cultured for a total of 16 hours before being washed with PBS. Cells were stained with live/dead (Invitrogen) for 3 minutes at room temperature and then incubated with the surface antibody cocktail for 30min at 4C. The surface antibody cocktail included: CD20 BV510, 2H7; CD3 BUV395, SK7; CD27 BUV737, L128; CXCR5 BB515, RF8B2 (all from BD Biosciences); CD4 BV605, RPA-T4; CD8 BV650, RPA-T8; and CD45RA PerCP-Cy5.5, HI100 (all from BioLegend). After fixation and permeabilization (BD CytoFix/CytoPerm) for 20 minutes at 4C, cells were incubated with the intracellular antibody cocktail (IFNg APC, B27; TNF BV421, Mab11; IL-2 PE, MQ1-17H12; all from BioLegend). Cells were washed in Perm/Wash buffer, resuspended in PBS+1%FCS, and acquired on a BD LSR Fortessa.
8
SARS-CoV-2 Spike Peptide-Specific T-cell Assay
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Cryopreserved human PBMC were thawed and rested for 4 h at 37 °C. Cells were cultured in 96-well plates at 1 × 106 cells/well and stimulated for 20 h with 2 μg/peptide/mL of peptide pools (15mer, overlapping by 11) covering the S1 or S2 domains of SARS-CoV-2. Selected donors were also stimulated with SEB (1 μg/mL) as a positive control, or individual peptides at 2 ug/mL: NCTFEYVSQPFLMDL (S1 epitope; previously described in ref. 45 (link)); LPIGINITRFQTLLA (S1 epitope); GWTFGAGAALQIPFA (S2 epitope); ALQIPFAMQMAYRFN (S2 epitope); LLQYGSFCTQLNRAL (S2 epitope;19 (link),45 (link)); QALNTLVKQLSSNFG (S2 epitope). Following stimulation, cells were washed, stained with Live/dead Blue viability dye (ThermoFisher), and a cocktail of monoclonal antibodies: CD27 BUV737 (L128), CD45RA PeCy7 (HI100), CD20 BUV805 (2H7), (BD Biosciences), CD3-BV510 (SK7), CD4 BV605 (RPA-T4), CD8 BV650 (RPA-T8), CD25 APC (BC96), OX-40 PerCP-Cy5.5 (ACT35), CD69 FITC (FN50), CD137 BV421 (4B4-1) (Biolegend), and CXCR5 PE (MU5UBEE, ThermoFisher). Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSR Fortessa using BD FACS Diva. Gating is shown in Supplementary Fig. 7 .
9
Flow Cytometric Analysis of Lung T-cell Subsets
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Cells used for in vitro or in vivo cytokine analysis were stimulated with PMA (20ng/mL; Sigma Aldrich), Ionomycin (1μg/mL; Sigma Aldrich), and Brefeldin A (2μg/mL; Sigma Aldrich) for 4 hours prior to flow staining (W. Lu et al., 2015 (link)). For intracellular staining, cells were fixed and permeabilized using the Mouse FOXP3 Buffer Set (BD) per the manufacturer’s protocol. The fluorophore-conjugated antibodies used in this study were as follows: Live/Dead Fix Blue (Invitrogen), CD3 PerCPCy5.5 (Biolegend), TCRb PE/Cy7 (Biolegend), CD4 PB (Biolegend), CD4 AF700 (Biolegend), CD8 BV650 (Biolegend), CD25 BV421 (Biolegend), FOXP3 AF488 (Biolegend), ROR gamma T PE (Invitrogen), TCF1 AF647 (Cell Signaling Technologies), TCF1 PE (Biolegend), IFNγ AF647 (Biolegend), IL17A FITC (Biolegend), IL17A PE (ebioscience). Representative flow cytometric gating and quantification strategy for detection of lung Th1/Th17 and Tc1/Tc17 cell populations is shown in Supplementary Figure 3 . Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).
10
Allogeneic Immune Response Assessment
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To test allogeneic immune responses, a novel mdMLR was used [27] . EV preparations to be tested (regularly 25 mg) were applied to 6x10 5 cells of a mixed peripheral blood mononuclear cell pool of 12 different donors and cultured in a final volume of 200 mL per well within 96-well u-bottom shape plates (Corning, Kaiserslautern, Germany) for 5 days. Thereafter, cells were harvested, stained with a collection of specifically selected fluorescent-labeled antibodies (CD4-BV785 [300554, Clone: RPA-T4, BioLegend], CD8-BV650 [344730, Clone: SC-1, BioLegend], CD25-PE [12-0259-42, Clone: BC-96, Thermo Fischer Scientific] and CD54-AF700 [A7-429-T100, Clone: 1H4, EXBIO]) and analyzed on a CytoFLEX flow cytometer (Software Cytexpert 2.3, Beckman-Coulter). Activated and non-activated CD4 T cells were discriminated by means of their CD25 and CD54 expression, respectively.
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