48 well plates (Corning) were coated with either 1.5 μg/mL anti-CD3 (Biolegend) and 5 μg/mL Isotype control antibody, 1.5 μg/mL anti-CD3 and 5 μg/mL PD-L1 (Biolegend), or 1.5 μg/mL anti-CD3 and 5 μg/mL PD-L2 (Biolegend) for 16 hours at 4°C. Mononuclear cells were isolated from whole blood of healthy donors by Lymphoprep density gradient (Stemcell). CD8 T cells were then purified by CD8 Microbeads (Miltenyi). Prior to stimulation, 5 x 105 cells per condition were stained with 2 μM CellTrace Far Red proliferation dye (Thermo) for 20 minutes at 37°C protected from light. Excess proliferation dye was neutralized with warm media, the stained cells were washed in PBS, resuspended in enriched media, and then added to the appropriate stimulation wells at 0.5 mL per well. Cells were incubated for 4 days at 37°C with 5% CO2. On day 4 the cells were analyzed for proliferation dye intensity and surface protein expression by flow cytometry as described below.
Anti cd3
Manufactured by BioLegend
Sourced in United States, United Kingdom, Germany, France, Panama
Anti-CD3 is a primary antibody that binds to the CD3 complex, a key component of the T cell receptor (TCR) signal transduction pathway. This antibody can be used for the detection and/or enrichment of T cells in various applications.
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Lab products found in correlation
Anti cd28, by BioLegend (142 mentions)
Anti cd4, by BioLegend (86 mentions)
Anti cd8, by BioLegend (73 mentions)
Anti cd45, by BioLegend (61 mentions)
Anti cd11b, by BioLegend (36 mentions)
Anti cd19, by BioLegend (30 mentions)
Anti ifn γ, by BioLegend (29 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (25 mentions)
Soluble anti cd28, by BioLegend (24 mentions)
Anti cd44, by BioLegend (22 mentions)
Anti cd11c, by BioLegend (22 mentions)
Anti il 4, by BioLegend (21 mentions)
Anti nk1, by BioLegend (16 mentions)
Anti f4 80, by BioLegend (16 mentions)
Anti cd62l, by BioLegend (14 mentions)
Celltrace violet, by Thermo Fisher Scientific (14 mentions)
Anti gr1, by BioLegend (13 mentions)
Anti ly6g, by BioLegend (13 mentions)
Anti pd 1, by BioLegend (13 mentions)
Anti tnf α, by BioLegend (13 mentions)
468 protocols using anti cd3
1
CD8+ T Cell Activation Assay
2
TMAO-Mediated T Cell Suppression Assay
3
Blocking PD-L1 on Tumor-Derived sEVs Enhances CD8+ T Cell Activation
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To block PD-L1 on sEV surface, the purified sEVs (200 μg) were incubated with PD-L1 blocking antibodies (10 μg/ml) or IgG isotype antibodies (10 μg/ml) in 100 μl PBS, and then rinsed with 30 ml PBS and pelleted by ultracentrifugation twice to remove the non-bound free antibodies. Human peripheral CD8+ T cells (1 × 105 per well-96 well plate) obtained from Human Immunology Core of University of Pennsylvania or murine CD8+ T cells (1 × 105 per well-96 well plate) purified from splenocytes and lymphocytes using EasyJet Mouse CD8+ T-cell Isolation Kit (STEMCELL) were stimulated with anti-CD3 (2 μg/ml, Biolegend) and anti-CD28 (2 μg/ml, Biolegend) antibodies for 24 hr and then incubated with indicated WM9 cell/xenograft-derived sEVs or B16F10 cell/xenograft-derived sEVs (20 μg/ml) with or without PD-L1 blocking for 48 hr in the presence of anti-CD3 and CD28 antibodies. The treated CD8+ cells were then collected, stained, and analyzed by flow cytometry. For BVD-treatment in vitro, indicated WM9 or B16F10 cell lines were pretreated with BVD-523 at 2 μM concentration for 24 hr before sEV collection.
4
Flow Cytometric Analysis of GC Tfh and B Cells
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Draining (inguinal) lymph nodes (dLNs) from immunized mice were harvested 10 days post-boost (DOL 24) as previously reported (25 (link), 52 (link), 53 (link)). To prepare a single-cell suspension, dLNs were pressed using the plunger end of a syringe. Then, cells were washed and stained with the following antibodies: for GC Tfh cells, anti-CD45, anti-B220, anti-CD3, anti-CD4, anti-programmed death-1 (CD279 or PD-1), anti-CXCR5; for GC B cells, anti-CD45, anti-B220, anti-CD3, anti-GL7, and anti-Syndecan-1 (CD138) (all from BioLegend). GC Tfh cells were defined as viable singlet CD45+ B220− CD3+ CD4+ CXCR5+ PD-1+ cells. GC B cells were defined as viable singlet CD45+ B220+ CD3− CD138− GL-7+. Cells were acquired on a BD LSRFortessa (BD Biosciences) and data were analyzed using FlowJo v.10 software (Tree Star). Absolute number of cell subsets were determined using CountBright Absolute Counting Beads (ThermoFisher Scientific). For a complete list of antibodies and fluorochromes used in the study, see Table S1 in Supplementary Material.
5
Hypertensive Kidney T Cell and DC Profiling
6
Murine T Cell Subsets Modulation
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Splenocytes from C57BL/6 mice were stimulated with anti-CD3 (1 μg/ml, BioLegend, USA) or anti-CD3 (1 μg/ml, BioLegend, USA) plus GITRL (1 μg/ml, MedChemExpress, China) for 48 h before harvesting. Th1, Th2, Th17 and Treg cells were determined by flow cytometry based on surface markers and intracellular cytokine expression. The culture supernatant was harvested for cytokine detection.
7
Liver Leukocyte Isolation and Flow Cytometric Analysis
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Leukocytes were isolated from liver as described previously52 (link). For flow cytometry, cell preparations were incubated with purified anti-CD16/CD32 antibody (BioLegend, San Diego, CA) for 15 min at 4 °C, and then stained with the following antibodies conjugated to fluorescent labels: anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, anti-CD19, anti-CD44, anti-CD62L, LAG3 and 2B4 (BioLegend).
For intracellular cytokine staining, approximately 106 liver mononuclear cells were stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience, California, USA) for 4 h, or EmP (5 μg/ml) for 10 h (37 °C, 5% CO2) as previously described52 (link). The cells were stained for surface anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, LAG3 and 2B4, and then stained for intracellular anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α, anti-IL-10 and granzyme B (Biolegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). ALSRFortessa flow cytometry (BD Immunocytometry Systems, San Jose, CA) was used to acquire data, which were analyzed with Flowjo software (Treestar, Inc., Ashland, OR).
For intracellular cytokine staining, approximately 106 liver mononuclear cells were stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience, California, USA) for 4 h, or EmP (5 μg/ml) for 10 h (37 °C, 5% CO2) as previously described52 (link). The cells were stained for surface anti-NK1.1, anti-CD3, anti-CD4, anti-CD8β, LAG3 and 2B4, and then stained for intracellular anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α, anti-IL-10 and granzyme B (Biolegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). ALSRFortessa flow cytometry (BD Immunocytometry Systems, San Jose, CA) was used to acquire data, which were analyzed with Flowjo software (Treestar, Inc., Ashland, OR).
8
Multiparameter Flow Cytometry Analysis of NK Cells
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The cells were incubated with Fc blockade reagent for 20 min at room temperature and stained with fluorochrome-conjugated antibodies in serum and sodium azide-containing buffer. Flow cytometry was performed as previously described (16 (link)). Antibodies in the present study were listed as follows: FITC anti-CD56 (Cat#362546, BioLegend, USA), PE anti-NKp30 (Cat#325208, BioLegend, USA), anti-CD56 (Cat#362508, BioLegend, USA), anti-NKp44 (Cat#325108, BioLegend, USA), anti-CD27 (Cat#356406, BioLegend, USA), PerCP-Cy5.5 anti-CD3 (Cat#317336, BioLegend, USA), anti-NKG2D (Cat#320818, BioLegend, USA), APC anti-NKp46 (Cat#331918, BioLegend, USA), anti-CD11b (Cat#301310, BioLegend, USA), anti-CXCR6 (Cat#356006, BioLegend, USA), anti-CD3 (Cat#317318, BioLegend, USA), PE-Cy7 anti-NKp46 (Cat#331916, BioLegend, USA), anti-CD3 (Cat#317334, BioLegend, USA), APC-Cy7 anti-CD3 (Cat#317342, BioLegend, USA), and anti-CD45 (Cat#304014, BioLegend, USA). FlowJo software was used for data analyzing (Tree Star, Inc., Ashland).
9
Immune Cell Profiling by Deep Sequencing
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T cells, B cells, and monocytes from P4 were sorted for deep sequencing further. PBMCs were stained with anti-CD3, anti-CD19, anti-CD14, and anti-CD16 antibodies (all from BioLegend, USA). Sorted cells were extracted using the Axygen DNA Mini Kit (Axygen, China). DNA sequencing was conducted as described above. PBMCs were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45 RA, anti-CD27, anti-CD19, anti-CD24, anti-IgD, anti-CD38, anti-CD16, anti-CD56, anti-β, and anti-γδ antibodies (all from BioLegend, USA).
10
Optimizing Nanovial Cell Loading for T Cell Studies
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Nanovials labeled with anti-CD45 antibodies were prepared using the procedures described above. To test cell concentration dependent loading of nanovials 0.15 × 106 (0.8 cells per nanovial), 0.3 × 106 (1.6 cells per nanovial), and 0.47 × 106 (2.4 cells per nanovial) of cell tracker deep red stained human primary T cells were each seeded onto 187,000 nanovials in a 24-well plate and recovered as described above. Loading efficiency was analyzed using a custom image analysis algorithm in MATLAB. The software measured the total number of nanovials in each image frame, then the number of cells in each nanovial was manually counted to record the total number of nanovials with 0, 1 or 2 or more cells (n > 2000). For comparing loading with different cell binding motifs, nanovials were labeled with 140 nM of each biotinylated antibody: anti-CD3 (Biolegend, 317320), anti-CD3 and anti-CD28 (Biolegend, 302904), or anti-CD45. Nanovials were seeded with 0.3 million cells in each well. To determine the effect of increased anti-CD45 concentration on nanovials, nanovials were labeled by incubating with 0, 70, 140, or 210 nM of anti-CD45 antibodies and seeded with 0.3 million cells in a 24-well plate. After cell binding and recovery of nanovials, the number of cells in each nanovial was analyzed using the same image analysis algorithms mentioned above (n > 2000).
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