NP conjugated Tregs were seeded in cRPMI at 1 × 106/ml. To some BSA-NP-coupled cultures, exogenous soluble IL-2 was added at 600 IU/ml as a positive control. On the indicated days, cells were stained for phosphorylated STAT5 (pSTAT5), using BD Biosciences Phosflow™ reagents and instructions. Briefly, cells were first stained with Live/Dead Near IR dye (Invitrogen), then CD25-BB515 (Clone: 2A3, BD Horizon™). Following washing, cells were fixed by BD Cytofix™ (BD Biosciences) and permeabilized with BD Perm buffer III (BD Biosciences). Cells were stained pSTAT5-Alexa Fluor® 647 (Clone: 47/Stat5 pY694, BD Biosciences).
Live dead near ir dye
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
The Live/Dead Near IR dye is a fluorescent labeling agent used in cell viability assays to distinguish live and dead cells. It is a near-infrared dye that can be detected using appropriate fluorescence instrumentation.
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Lab products found in correlation
Easysep, by STEMCELL (4 mentions)
Dynabead, by Thermo Fisher Scientific (4 mentions)
Cytofix, by BD (3 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (3 mentions)
Z2 coulter counter, by Beckman Coulter (3 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)
Lsrfortessa, by BD (3 mentions)
Pstat5 alexa fluor 647, by BD (2 mentions)
Perm buffer 3, by BD (2 mentions)
Phosflow, by BD (2 mentions)
Anti cd3 okt3, by BioLegend (2 mentions)
Facsaria 3, by BD (2 mentions)
Facsaria 2 flow cytometer, by BD (2 mentions)
Nylon cell strainer, by Corning (2 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Anti cd69 fn50, by BioLegend (1 mentions)
Anti tcr β ip26, by BioLegend (1 mentions)
Anti cd137 4b4 1, by BD (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Rosettesep human t cell enrichment cocktail, by STEMCELL (1 mentions)
21 protocols using live dead near ir dye
1
Phosphorylated STAT5 Quantification in NP-Tregs
2
T Cell Activation and Antigen Presentation
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T cells were isolated from a leukoreduction system chamber from an HLA-A∗02 positive healthy donor from the Stanford institutional blood bank using the RosetteSep human T cell enrichment cocktail (Stem Cell Technologies) and viably stored in liquid nitrogen. For T cell activation, T cells were thawed and stimulated with anti-CD3/CD28 beads (Life Technologies) in the presence of IL-2 (100 IU/mL). On days 1 and 2, activated T cells were retrovirally transduced using Retronectin (Takara) coated plates in media containing 100 IU/mL IL-2. anti-CD3/CD28 beads were removed on day 3 and media containing IL-2 were replenished once every 2 days. Following 8 days of in vitro expansion, T cells were co-cultured with 293A2 cells expressing full-length TMEM161A, EntS, LMP2, FluM1, or GFP alone at a 1:1 ratio. Following 18 h incubation, cells were stained with anti-CD3 (OKT3, Biolegend), anti-CD69 (FN50, Biolegend), anti-TCR⍺/β (IP26, Biolegend), anti-CD137 (4B4-1, BD Biosciences), and live/dead near-IR dye (Invitrogen). Data were acquired using FACS Fortessa (BD Biosciences) automated high throughput sampler and analyzed using FlowJo software (Treestar).
3
NK Cell-Mediated Cytotoxicity Assay
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The NK-sensitive K562 erythroleukemia target cells were labeled by the cell tracer dye eFluor 450 (eBioscience) 12 h prior to the cytotoxicity assay [9 (link)]. The lentiviral-transduced NK-92 cells and labeled K562 target cells were mixed together and stained with Live/Dead near-IR dye (Invitrogen) and analyzed by FCM, as described previously [8 (link)]. Anti-human CD56-PE-Cy7 and CD107a-PE monoclonal antibodies (mAbs) were purchased from eBioscience (San Diego, CA, USA). For degranulation assays, the transduced NK-92 cells were mixed with K562 cells, and incubated in the presence of fluorochrome-conjugated anti-CD107a Ab; K562 target cells served as a stimulus for NK-92 cell degranulation [8 (link)]. To identify NK-92 effector cells, cells were stained with fluorochrome-conjugated anti-CD56 mAb after incubation with K562 stimulator cells and CD107a cell surface expression on NK-92 cells was detected by FCM as previously described [10 (link)].
4
Rat Immune Cell Flow Cytometry
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The following rat reactive antibodies (clone, staining dilution) were obtained from BD Biosciences, eBioscience, and Miltenyi Biotec for use in flow cytometric analysis: CD3-VioGreen (REA223, 1:300), CD3-BV421 (1F4, 1:200), CD4-PerCP-eFluor710 (OX35, 1:250), CD8-BV786 (OX8, 1:100), IFNγ-AF647 (DB-1, 1:100), IFNγ-eFluor660 (DB-1, 1:50), TNFα-FITC (TN3-19.12, 1:50), and TNFα-PE (TN3-19.12, 1:100). Surface stains were performed for 20 min at 4° C in 50 μL PBS supplemented with 2.5% FBS (Gibco) and 0.1% sodium azide (Sigma Aldrich) in 96-well U-bottom plate. Dead cells were excluded from analysis using the LIVE/DEAD nearIR dye (Invitrogen) per manufacturer’s protocol. Events were collected on a BD Fortessa flow cytometer following compensation with UltraComp eBeads (Invitrogen). Data were analyzed using FlowJo v7.6.5 (Tree Star).
5
Treg pSTAT5 Activation Assay
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NP conjugated Tregs were seeded in cRPMI at 1 × 106/ml. To some BSA‐NP‐coupled cultures, exogenous soluble IL‐2 was added at 600 IU/ml as a positive control. On the indicated days, cells were stained for phosphorylated STAT5 (pSTAT5), using BD Biosciences Phosflow™ reagents and instructions. Briefly, cells were first stained with Live/Dead Near IR dye (Invitrogen), then CD25‐BB515 (Clone: 2A3, BD Horizon™). Following washing, cells were fixed by BD Cytofix™ (BD Biosciences) and permeabilized with BD Perm buffer III (BD Biosciences). Cells were stained pSTAT5‐Alexa Fluor® 647 (Clone: 47/Stat5 pY694, BD Biosciences).
6
HLA-A*02 Tetramer Staining Protocol
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Recombinant HLA-A∗02 monomer with UV exchangeable peptide were either synthesized as previously described (Altman and Davis, 2003 ) or purchased commercially (Biolegend). UV peptide exchange was performed over 20 min with 1 mM of peptide in PBS using Strategene UV Stratalinker 2400. Streptavidin conjugated fluorophore was added incrementally the following day for a final 4:1 molar ratio of MHC:streptavidin. Tetramer staining was performed in PBS plus 2% FBS in Fc Blocking solution (Biolegend) at room temperature for 1 h. For peripheral blood samples, cells were subsequently stained with anti-TCRγδ (B1, Biolegend), anti-CD19 (H1B19, Biolegend), anti-CD14 (M5E2, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD4 (RPA-T4, Biolegend), anti-CD8 (HIT8a, Biolegend), and live/dead near-IR dye (Invitrogen). For tumor samples, cells were stained with anti-CD4 (OKT4, Biolegend), anti-CD8 (HIT8a, Biolegend), anti-CD3 (UCHT1, Biolegend), anti-CD45 (H130, Biolegend).
7
Detecting Virus-Specific CD8+ T Cells
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Biotinylinated rat RT1-Al monomers specific for the RHV E1191 and NS5B2511 epitopes were obtained from the NIH Tetramer Core Facility and tetramerized with streptavidin-APC (Prozyme) via standard protocol. For direct detection of virus-specific CD8 T cell populations, liver-infiltrating leukocytes were stained for 30 min at 4° C (1:100) with tetramer alone followed by surface labeling with anti-rat CD3 and CD8 as above. Following staining with LIVE/DEAD near IR dye (Invitrogen), cells were fixed in 1% PFA prior to analysis.
8
SARS-CoV-2 Spike Protein T-cell Response
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Cryopreserved peripheral blood mononuclear cells (PBMC) were thawed and rested overnight. PBMC (1 × 10 6 per well) were stimulated with a SARS-CoV-2 spike overlapping peptide pool (JPT, 1 μg/mL each peptide; 0.4% final DMSO concentration), 0.4% DMSO as a negative control, or PHA-P (Remel, Lenexa, KS, USA; 1.6 μg/mL final) as a positive control, in the presence of anti-CD28 (1:1000, L293, BD Biosciences) and anti-CD49d (1:1000, L25, BD Biosciences) antibodies at 37°C for 6 hours. Brefeldin A (Sigma) was added after 2 hours. Cells were stained with Live/Dead Near IR dye (1:250, Invitrogen), treated with FACS lyse (BD Biosciences) and frozen at -80°C. For staining, the cells were thawed, washed, and permeabilized with Permeabilizing solution 2 (BD Biosciences) then stained with fluorochrome labeled monoclonal antibodies to anti-human CD3-PE-Texas Red (1:40, UCHT, Beckman Coulter), anti-human CD4-BV510 (1:83, SK3, Biolegend), anti-human CD8-PerCP-Cy5.5 (1:33, SK1, BD Biosciences), and the activation markers anti-human CD40L-BV421 (1:167, TRAP1, BD Biosciences), anti-human IFNγ-PE (1:500, 4S.B3, BD Biosciences), anti-human IL2-APC (1:250, MQ1-17H12, BD Biosciences), and anti-human TNFα-FITC (1:400, Mab11, BD Biosciences). Events were recorded with BD Fortessa and analyzed with FlowJo (v10 for Mac; BD).
9
Inflammatory Cell Profiling in Murine Peritoneum
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Mice aged 8–12 weeks were injected intraperitoneally with 200 µl of PBS solution containing or not 0.5 µg of SF-IL-17A. After 4 h, mice were anesthetized with a mixture of 8 mg ml–1 of ketamine (Narketan) and 2 mg ml–1 of xylazine (Rometar). Anesthetized mice were beheaded to bleed out and peritoneal lavage was performed using 11 ml of PBS. Cell suspensions were stained with LIVE/DEAD near-IR dye (Life Technologies) and the mixture of primary antibodies (CD45.2, CD11b, F4/80, Ly6G, Ly6C) on ice and analyzed by flow cytometry using Cytek Aurora. CD45.2+ cells were separated into the following subsets: residual macrophages (CD11b+, F4/80+), neutrophils (F4/80−, Ly6G+, CD11b+) and inflammatory monocytes (F4/80−, Ly6G−, CD11b+, Ly6C+).
10
Detailed Protocol for Multiparameter Flow Cytometry Analysis
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For the surface staining, cells were incubated with diluted antibodies in PBS/0.5% gelatin or PBS/2% goat serum on ice. LIVE/DEAD near‐IR dye (Life Technologies) or Hoechst 33258 (Life Technologies) was used for discrimination of live and dead cells. For the intracellular staining, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience, 00‐5523‐00). For some experiments, enrichment of CD8+ T cells was performed using magnetic bead separation kits EasySep (STEMCELL Technologies) or Dynabeads (Thermo Fisher Scientific) according to manufacturer's instructions prior to the analysis or sorting by flow cytometry. Cells were counted using Z2 Coulter Counter (Beckman) or using AccuCheck counting beads (Thermo Fisher Scientific) and a flow cytometer. Flow cytometry was carried out with a FACSCantoII, LSRII, or a LSRFortessa (BD Bioscience). Cell sorting was performed using a FACSAria III or Influx (BD Bioscience). Data were analyzed using FlowJo software (TreeStar).
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