Anti-CD31 (390), mAbs from ThermoFisher; PB- & BV711-anti-mouse CD45 (30-F11 dilution 1/500), AF488-anti-CD115 (AFS98 dilution 1/300), PE-Cy7-anti-CD31 (390 dilution 1/300), AF488- & AF647-anti-CD54 (YN1/1.7.4 dilution 1/300), APC-anti-CD140b (18A2 dilution 1/300), APC-Cy7-anti-CD115 (AFS98 dilution 1/300), AF647- & PE-Cy7-anti-CD117 (2B8 dilution 1/300), PB-FcεRI (MAR1 dilution 1/300), AF647-anti-IL-17A (TC11-18H10.1 dilution 1/300), AF594-anti-CD4 (GK1.5 dilution 1/300), AF700-anti-CD3 (17A2 dilution 1/300), BV605-anti-CD41 (MWReg30 dilution 1/300), PE-anti-CD49d (R1-2 dilution 1/300), AF647-anti-CD11c (N418 dilution 1/300), Rat IgG1 mAbs from Biolegend; Anti-IL-17RA (G-9 dilution 1/100) from SantaCruz; Desmin (D33 dilution 1/100) were obtained from Dako; Anti- αSMA (1A4 dilution 1/300) from Sigma-Aldrich; anti-mouse CXCL1 (polyclonal dilution 1/100) from R&D systems. Anti-mMCP-1 (RF6.1 dilution 1/100) from ThermoFisher. Anti-MRP14 mAb (dilution 1/500) was a gift form Dr Nancy Hogg (The Francis Crick Institute, UK).
Anti cd3 af700
Manufactured by BioLegend
Sourced in United States
Anti-CD3-AF700 is a fluorescently-labeled antibody that binds to the CD3 complex, a key component of the T cell receptor. This product is intended for use in flow cytometry applications to identify and characterize T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Perm wash, by BD (3 mentions)
Lsrfortessa, by BD (2 mentions)
Live dead fixable red dead cell stain, by Thermo Fisher Scientific (2 mentions)
Anti cd4 pb, by BioLegend (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Zombie aqua vital dye, by BioLegend (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Fix perm solution, by BD (2 mentions)
Anti cd8 fitc, by BD (2 mentions)
Anti cd4 bv605, by BioLegend (2 mentions)
Anti fc apc, by BioLegend (2 mentions)
Perm wash, by Beckman Coulter (2 mentions)
Anti gag kc 57 rd1, by Beckman Coulter (2 mentions)
Nonspecific igg, by BioLegend (2 mentions)
Live dead blue, by Thermo Fisher Scientific (2 mentions)
Anti cd4 bv650, by BioLegend (2 mentions)
Desmin, by Agilent Technologies (1 mentions)
Anti cd31 390, by Thermo Fisher Scientific (1 mentions)
Anti cd49d pe, by BioLegend (1 mentions)
Anti il 17ra, by Santa Cruz Biotechnology (1 mentions)
15 protocols using anti cd3 af700
1
Comprehensive Multicolor Flow Cytometry Panel
2
Brain Tissue Cell Isolation and Characterization
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Cells were isolated from brain tissue as previously described [20 (link)], with some modifications. Briefly, isolated tissues were cut up into fine pieces and were dissociated in buffer containing HBSS (without calcium/magnesium), 5% FBS, 10 μM HEPES, 2 mg/mL collagenase D (Sigma-Aldrich) and 28U/mL DNaseI (NEB) at 37 °C for 45 min. Every 15 min, dissociated tissue was pipetted up and down using sequentially smaller Pasteur pipettes to homogenize tissue debris. Homogenate was filtered through a 70-μm filter and centrifuged 10 min at 300 g. Pellets were resuspended in PBS and stained with an APC-Cy7 LIVE/DEAD™ fixable dead cell stain kit (Invitrogen). Cells were washed, resuspended in FACS buffer with Fc block (BD Biosciences) and stained with the following antibodies: AF700 anti-CD3 (BioLegend, clone:17A2), BV711 anti-Ly6C (BioLegend, clone: 1A8), BV421 anti-Ly6G (BioLegend, clone: HK1.4), FITC anti-CX3CR1 (BioLegend, clone: SA011F11), BV510 anti-CD19 (BioLegend, clone: 6D5), PerCPCy5.5 anti-CD45 (BioLegend, clone: 30-F11), and APC anti-Cd11b (BioLegend, clone: M1/70). Samples were measured on an LSRFortessa (BD Biosciences) and analyzed using FlowJo software.
3
Fluorescent Labeling of Human IgA for Leukocyte Binding
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Human IgA purified from post OIT sera was fluorescently labeled using the Pierce FITC antibody labeling kit (Thermofisher Scientific, Rockford, IL) according to the manufacturer’s instructions. For IgA binding experiments, donor whole blood was stained with labeled IgA, along with antibodies to human leukocyte markers PE anti-CCR3, APC anti-FcεR1, AF700 anti-CD3, PE-Cy7 anti-CD16, BV510 anti-CD14 all from (Biolegend, San Diego, CA) for 30 minutes at 37°C.
4
Comprehensive Immunophenotyping of Immune Cells
5
Phenotypic analysis of PBMCs
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Peripheral blood mononuclear cells at 1 × 106 viable cells/mL (final concentration) in complete DMEM/F12 were incubated with anti-CD3 AF700 (Biolegend), anti-CD28 Pacific blue (PB) (Biolegend), anti-CD161 PE-Cy7 (Biolegend), anti-CD158d PE (Biolegend), and anti-TCR Vα24-Jα18 FITC (Biolegend) or the corresponding isotype controls. All antibodies were purchased from Biolegend (United Kingdom). After 20 min at 20°C cells were washed, and fixed with 1% formalin. Data were acquired by flow cytometry (EC800 Sony) and analyzed using FlowJo software.
6
Comprehensive PBMC Immunophenotyping Panel
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PBMC were washed and stained with Live/Dead fixable red dead cell stain (Invitrogen) followed by surface staining with the following antibodies: anti-CD3-AF700, anti-CD4-PB, anti-CD14-PE/Cy7, anti-CD16-AF488, anti-CD19-PE/Cy5, anti-CD25-APC/Cy7, γδ T cell receptor (TCR)-APC, anti-cytotoxic-T-lymphocyte-associated antigen (CTLA)-PE (all from BioLegend), anti-CD8-efluorNC605, and anti-CD127-efluorNC650 (eBioscience). Fluorescence minus one (FMO) controls were used to identify boundaries of gates for CD25, γδTCR, CD127, and CTLA. Samples were acquired on a BD LSR II flow cytometer. Results are presented as percentages of cells after gating out of dead cells and doublets. CD4+ and CD8+ T cells were identified as CD3+ cells, while CD14+/− and CD16+/− cells were identified as CD3− and CD19− populations. CTLA+ and CD25+ CD27− populations were gated on the CD4+ cells.
7
Quantifying T-cell Retroviral Transduction
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We employed biotin-SP (long spacer) AffiniPure F(ab′)2 Fragment Goat Anti-Mouse IgG, F(ab′)2 fragment specific (Jackson Immunoresearch, West Grove, PA, USA) to assess the transduction efficiency of PBMCs. The cells were stained with these antibodies at a 1:1000 dilution and incubated for 20 min in the dark at room temperature. After two washes, we added streptavidin-PE at a dilution of 1:600, along with monoclonal antibodies targeting surface markers (anti-CD3-AF700 (Biolegend, USA)) and Zombie Aqua vital dye (Biolegend, USA) to evaluate the culture’s viability. Staining was conducted for 20 min at room temperature in the dark. The stained cells were then washed with PBS containing 0.1% NaN3 and subjected to analysis using an Attune NxT flow cytometer (Thermo Fisher, Waltham, MA, USA). We gated cells from debris, singlets from the cells, alive cells from the singlets, CD3-positive cells from the alive cells, and anti-Fab-positive cells from the CD3-positive cells. We observed 50 ± 12.28% retroviral transduction of said T-cells (mean ± standard deviation), with a minimum of 37.7% and a maximum of 62.2% (Scheme 2 ).
8
T Cell Proliferation Assay for PBMCs
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PBMC were counted and plated in 24-well plates at 1 × 106 PBMC/1 ml R10. Plates were incubated for 6 days at 37°C and 5% CO2 with either 2 μg/ml PPD or 1 μg/ml/peptide of a single pool of antigen 85A peptides. Two wells of PBMC were left unstimulated in R10. After 3 days, PHA was added to one of the unstimulated wells at 0.3 μg/ml. Three days later, the cells were washed in phosphate-buffered saline (PBS) and stained with amine-reactive viability dye (Live/Dead fixable red dead cell stain; Invitrogen). Cells were then permeabilized with Perm/Wash (BD Biosciences) and incubated with monoclonal antibodies: anti-CD3-AF700, anti-CD4-PB, anti-Ki67-phycoerythrin (PE) (BioLegend), and anti-CD8-allophycocyanin (APC)/AF750 (Beckman Coulter). Cells were then washed and acquired on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (Tree Star Inc.). Dead cells were excluded from the analysis, while singlet CD3+ T cells, CD3+ CD4+ T cells, and CD3+ CD8+ T cells were included. Proliferating cells are presented as the percentage of Ki67+ T cells out of the total CD4+ or CD8+ T cells. Background (unstimulated) values were subtracted from all data.
9
Assessing Retroviral Transduction Efficiency in T Cells
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We employed MHC-biotin tetramers specific to the studied NY-ESO-1 TCR, conjugated with streptavidin-PE to assess the transduction efficiency of PBMCs. The cells were stained with these tetramers at a 1:100 dilution and incubated for 20 min in the dark at room temperature. The MHC tetramers were provided by Prof. H. Shiku (Mie University Graduate School of Medicine, Mie, Japan). After two washes, we added streptavidin-PE (Biolegend, San Diego, CA, USA), at a dilution of 1:600, along with monoclonal antibodies targeting surface markers (anti-CD3-AF700 (Biolegend, San Diego, CA, USA)) and Zombie Aqua vital dye (Biolegend, San Diego, CA, USA) to evaluate the culture’s viability. Staining was conducted for 20 min at room temperature in the dark. The stained cells were then washed with PBS containing 0.1% NaN3 and subjected to analysis using an Attune NxT flow cytometer (Thermo Fisher, Waltham, MA, USA). We gated cells from debris, singlets from the cells, alive cells from the singlets, CD3-positive cells from the alive cells, and MHC tetramer-positive cells from the CD3-positive cells (Figure 2 ). We observed a 22.13 ± 7.11% retroviral transduction of said T cells (mean ± standard deviation, n = 6), with a minimum value of 13.40% and a maximum of 33.5%.
10
Antibody-Mediated Recognition of Env-Expressing Cells
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Infected cells were washed twice in PBS, and aliquoted into 96 well plates (2×105 cells/well for PBMCs, 5×104 for cell lines). Cells were then pelleted and resuspended in PBS containing 25 μg/ml of the appropriate anti-Env antibody, and incubated for 60 minutes at RT. The plate was then washed twice with PBS, and extracellular staining was performed with Anti-CD3-AF700 (Biolegend clone SK7), Anti-CD4-BV605 (Biolegend clone OKT4), anti-CD8 FITC (BD clone HIT8a), anti-Fc APC (Biolegend clone HP6017), and Live/Dead Blue (ThermoFisher) and incubated for 20 minutes at RT. Samples were then washed twice and cells were fixed with Fix/Perm solution (BD), washed twice with Perm/Wash (BD) and resuspended in Perm/Wash containing anti-gag KC-57-RD1 (Beckman) and incubated for 20 minutes. Cells were then washed twice with Perm/Wash and resuspended in PBS for data acquisition by flow cytometry. To avoid potential differences in env expression, all HC antibodies and controls were compared for recognition on the same batch of infected cells at the same time. Infected-cell recognition rates among each virus were Z-scored using the standardize function in Microsoft Excel. Negative controls include nonspecific IgG (Biolegend), IVIG, and PBS conditions. Positive controls include polyclonal HIVIG, and combinations of bNAbs directed against multiple binding sites (such as 10E8, PGT121, and 3BNC117).
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