Pullulan was purchased from Meihua Group (Hebei, China). PAA (average MW of 450,000) was purchased from Aladdin Industrial Corporation Chemical (Shanghai). N-(3-Dimethylaminopropyl)-N’-Ethylcarbodiimide Hydrochloride (EDC) was purchased from Heowns Chemical (Tianjin, China). 4-Dimethylaminopyridine (DMAP) and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd. Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. aCD47 (Catalog no. BE0270) used in vivo was purchased from BioX Cell. AF790-labeled goat anti-mouse IgG (H&L) (Catalog no. 115-655-146) was purchased from Jackson ImmunoResear. Anti-CD3-PerCP-Cy5.5 (Catalog no. 551163), anti-CD4-APC (Catalog no. 553051), anti-CD8-FITC (Catalog no. 553030), anti-CD86-PE (Catalog no. 553692), anti-CD80-APC (Catalog no. 560016), anti-Foxp3-PE (Catalog no. 563101), anti-CD11c-FITC (Catalog no. 557400), anti-CD62L-BV421 (Catalog no. 562910), anti-CD44-APC (Catalog no. 559250) and anti-CD11b-PE-Cy7 (Catalog no. 552850) were purchased from BD Biosciences. Anti-mouse CD206-PE (Catalog no. 141705) and anti-mouse F4/80-FITC (Catalog no. 123107) were purchased from Biolegend. Human IgG ELISA Quantitation Set (Catalog no. E80-104) was purchased from Bethyl Laboratories, Inc.
Anti cd3 percp cy5
Manufactured by BD
Sourced in United States
Anti-CD3-PerCP-Cy5.5 is a fluorochrome-conjugated antibody that binds to the CD3 antigen. CD3 is a complex of membrane proteins that is involved in activating T cells. The PerCP-Cy5.5 fluorochrome is used for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45 apc, by BD (3 mentions)
Live dead yellow, by Thermo Fisher Scientific (3 mentions)
Anti cd16 cd32 fc block, by BD (3 mentions)
Anti cd8 pe texas red, by Thermo Fisher Scientific (3 mentions)
Anti cd4 fitc, by BD (3 mentions)
Anti cd56 apc, by BD (3 mentions)
Anti cd25 pe, by BD (3 mentions)
Anti cd4 apc, by BD (2 mentions)
Anti foxp3 pe, by BD (2 mentions)
Anti cd11c fitc, by BD (2 mentions)
Anti cd14 fitc, by BD (2 mentions)
Anti il 10 apc, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Anti cd8 apc cy7, by BD (2 mentions)
Anti cd4 pacific blue, by Thermo Fisher Scientific (2 mentions)
Cytofix, by BD (2 mentions)
Anti cd19 apc, by BD (2 mentions)
Anti cd14 apc h7, by BD (2 mentions)
Anti cd8 pe, by BD (2 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (2 mentions)
31 protocols using anti cd3 percp cy5
1
Pullulan-based Immunotherapeutic Delivery
2
Competitive Binding of Monalizumab to HLA-E Tetramers
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Solution of biotinylated-HLA-E monomers (5nM solution, kindly provided by Novo Nordisk) was slowly mixed with PE-conjugated streptavidin (Southern Biotechnology) at a ratio 1/4 and then kept at 4°C until use. Competition of monalizumab and PE-conjugated tetramers was performed on peripheral blood from healthy donors (EFS). 100μL of whole blood was incubated for 1 h at 37°C with 50μL PBS containing PE-HLA-E tetramers (0.1μL) and monalizumab (ranging from 30μg/mL to 0,001μg/mL, 1/3 dilution) and then washed with PBS. Peripheral blood cells were then stained with anti-CD3-PercpCy5.5 (BD Biosciences), anti-CD45-APC (BD Biosciences), anti-CD3-PC7 (BD Biosciences) and anti-human IgG4 Fc-FITC (HP6025, Southern Biotechnology). Red blood cells were lyzed with Optilyse C solution (Beckman Coulter). Acquisition was performed on a FACSCanto II flow cytometer.
3
Quantification of RHAMM-specific CD8+ T cells
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Eight weeks after starting biweekly intradermal vaccination (i.e., after 4 vaccines) into the upper arms near the axilla, a DTH skin test was performed on the upper back with single intradermal injections of the vaccine or its components, including KLH alone. Two days later skin reactions were measured and skin biopsies were taken. DTH skin test-infiltrating lymphocytes (DILs) were collected and allowed to expand for 2-3 weeks in medium with 100 IU/mL of IL-2. After harvest, RHAMM-specific CD8+ T cells were quantified in DILs through flow cytometry following staining with R3 or R5 peptide-loaded (p)HLA-A*0201 tetramers (Fred Hutchinson Cancer Research Centre Seattle). For each condition, 1 × 106 viable thawed DILs were washed and stained for 30 min at 4°C with allophycocyanin-labeled pHLA-A*0201 tetramers. Samples were then stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain (cat# L34957, Thermo Fisher Scientific), anti-CD3-PerCP-Cy5.5 (cat# 560835, BD Biosciences), anti-CD14-FITC (cat# 345784 BD Biosciences), anti-CD19-FITC (cat# 345776, BD Biosciences) and anti-CD8-Pacific Blue (cat# MHCD0828, Thermo Fisher Scientific) for another 30 min at 4°C, followed by detection on a Cyflow ML flow cytometer (Partec). Dead (LIVE/DEAD+) cells and monocytes and B cells (FITC+) were excluded from analysis.
4
Induction of Regulatory T Cells
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The induction of different populations of Treg was determined following coculture of autologous PBL, stimulated with MOG- and MBP-derived peptides in the presence or absence of DC, as described above. At day 6, 10 µg/mL brefeldin A (GolgiStop, BD Pharmingen) was added to the DC/T cell coculture and incubated overnight at 37°C. Next, cells were harvested and membrane markers were stained with the following mouse anti-human monoclonal antibodies: anti-CD3-PerCP-Cy5.5 (BD Biosciences), anti-CD4-allophycocyanin-H7 (anti-CD4-APC-H7) (BD Biosciences), anti-CD8-Pacific Blue (Life Technologies), and anti-CD25-PE-Cy7 (BD Biosciences). Subsequently, cells were fixed and permeabilized using a FOXP3 Staining Buffer Kit (eBioscience, Hatfield, UK), according to manufacturer's instructions, and intracellular markers were stained with anti-FOXP3-alexa488 (BD Pharmingen), anti-TGF-β-PE (IQ Products, Groningen, Netherlands), and anti-IL-10-APC (BD Pharmingen). Labeled cells were analyzed on a Cyflow ML flow cytometer (Partec, Münster, Germany). For analytical flow cytometry, at least 5 × 104 CD3+ CD4+ CD8− lymphocytes were acquired. All results were analyzed using FlowJo software.
5
Flow Cytometry Analysis of Antigen-Specific T Cells
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For stimulated cells, 106 cells/well were suspended in 100 μl MEM (Cellgro, Manassas, VA)/5% FBS medium containing anti-CD107a-eFluor450 (LAMP-1) (eBiosciences, San Diego, CA), 1 μg/mL each of anti-CD28 and -CD49d (BD Biosciences) and 0.7 μg/ mL monensin (BD Biosciences), with or without peptides: 2.5 μg/mL each of NP147-155, NP55-69, and M2e2-24 or 2.5 μg/mL SARS M for 5–6 hours. After incubation, cells were washed and stained with Live/Dead fixable blue viability stain Vivid for UV excitation (Invitrogen). Mouse CD16/32-specific monoclonal antibody 2.4G2 was added to cells before incubation with anti-CD3-PerCp-Cy5.5, anti-CD8-APC-Cy7, anti-CD4-AF700 (all from BD Biosciences), anti-CD107a, and NP147–155-H2-Kd Tetramer-APC. Following washing, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences), and then incubated with anti-CD3, anti-CD8, anti-CD4 antibodies labeled as explained above. 30,000 events were acquired on a Fortessa flow cytometer (BD Biosciences,).
6
CD4+ T cell depletion protocol
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To deplete effector CD4+ T cells, mice were vaccinated on day 0, injected twice i.p. with 100 μg of a rat anti-CD4 mAb (clone GK1.5; Areta International) or isotype-matched control antibody (isot. ctr., rat IgG2b; R&D System) on day 6 and 8, and challenged with S. aureus on day 10. CD4+ cell depletion efficiency was evaluated by flow cytometry on heparinized blood taken the day before infection (day 9). For this purpose, RBCs were lysed with RBCs lysis buffer (Biolegend). White blood cells were stained with Live/Dead Yellow (Invitrogen), incubated with anti-CD16/CD32 Fc block (BD Biosciences), and stained with: anti-CD3-PerCP Cy5.5, (BD Pharmingen), anti-CD8-PE TexasRed (Invitrogen), and anti-CD4-Pacific Blue (Invitrogen) in PBS 0.1% BSA for 20 min at room temperature. Cells were fixed with Cytofix (BD Biosciences), suspended in PBS and analyzed by LSR II flow cytometer (BD Biosciences). No CD4+ T cells were detected in the peripheral blood indicating that depletion occurred efficiently (S2 Fig ).
7
Isolation and Purification of PBMC Subsets
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Isolation of PBMCs, T cells, B cells and monocytes Whole blood (10 ml) was collected in EDTA collection tubes from each subject, and PBMCs were isolated by density-gradient centrifugation with Ficoll-Paque Premium (GE Healthcate), according to the instructions. For the subsets of PBMCs isolation, the fresh PBMCs were incubated for 15 min at 4°C with flurescentconjugated monoclonal antibodies: anti-CD3-PerCP-Cy5.5, anti- CD14-PE, anti-CD19-APC (all from BD Biosciences). Stained cells were sorted on a BD FACSAria III (BD Biosciences). T cells were identified as CD3t/CD19-. Monocytes were isolated if cells were CD14t/CD3-. B cells were collected if cells were CD19t/CD3-. The stained cells were sorted to >98% purity.
8
Comprehensive Immune Cell Profiling by Flow Cytometry
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NK, T and Monocyte cell surface antigens were analysed by flow cytometry. Briefly, two million PBMCs were stained with a surface antibody’s cocktail: anti-CD3 PerCP-Cy 5.5, anti-CD56 APC and anti-CD16 PE-CyTM7 from BD Biosciences, anti-CD45 Krome Orange from Beckman Coulter and anti-Vδ2 Brilliant Violet 605 from Biolegend (Mix NK and gammadelta T cells); anti-CD4 FITC, anti-CD8 PE and anti-CD3 Pacific Blue from BD Biosciences and anti-CD45 Krome Orange (Mix T cells); anti-CD14 APC-H7 and anti-CD16 V450 from BD Biosciences and anti-CD45 Krome Orange (Mix Monocytes), in 1× PBS, 1% Bovine Serum Albumin (BSA) (from Sigma Aldrich) and 0.1% Sodium azide (NaN3) (from Serva) solution for 15 min at 4°C. Afterwards, PBMCs were washed with 0.3 mL of 1× PBS, 1% BSA and 0.1% NaN3 solution and centrifuged at 1600 rpm for 5 min. Next, PBMCs were fixed with 1% Paraformaldehyde (PFA) (Bio-Rad, Hercules, California, USA) in 1× PBS, 1% BSA and 0.1% NaN3 solution for 15 min at room temperature in the dark, washed as before and centrifuged at 1600 rpm for 5 min. Cells were acquired using a FACSLyric (BD Biosciences) and analysed by BD FACSuite software (BD Biosciences).
9
Immunophenotyping of Granulocytic Myeloid-Derived Suppressor Cells
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All antibodies were purchased from Biolegend (San Diego, CA, USA), except for anti‐CD14‐APCH7, anti‐CD3‐PerCPCy5.5, anti‐CD107a‐FITC and anti‐IFN‐γ‐PECy7, which were purchased from BD Biosciences (Franklin Lakes, NJ, USA). To determine the frequencies of gMDSCs, peripheral blood mononuclear cells were first stained with live/dead‐BV510 (Life Technologies, Waltham, MA, USA) and surface antibodies and then incubated for 20 minutes at 4°C in the dark. gMDSCs were counted as CD15+HLA−DR−CD14− cells derived from CD33+CD11b+ myeloid cells. For intracellular staining, cells were permeabilized with the Cytofix/Cytoperm Solution Kit (BD Bioscience) and stained with anti‐arginase I‐FITC (R&D, Minneapolis, MN, USA) or anti‐IFN‐γ‐PECy7; a matched isotype antibody was used as a negative control. After staining, cells were fixed in 1% paraformaldehyde and analysed using a FACS Verse flow cytometer and FlowJo software. For morphologic analysis, flow cytometric‐isolated gMDSCs were stained with a Wright‐Giemsa kit (Solarbio, Beijing, China) following the manufacturer's instructions.
10
Treg Suppressive Function Assessment
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To assess Treg suppressive function, isolated PBMCs were cultured alone or with MAPC cells at a 2:1 PBMC: MAPC cell ratio for 7 days as described above. At day 7, Tregs were isolated using EasySep™ Human CD4 + CD127LowCD25 + Regulatory T cell isolation kit (STEMCELL Technologies, Cambridge, MA) following manufacturer's instructions. Over 85% of the cells isolated were Tregs based on their phenotypic pro le (CD3 + CD4 + CD25 + , FoxP3 + , and CD127 low ) assessed by ow cytometry. Isolated Tregs were co-cultured with CellTrace Violet stain (ThermoFisher) labeled autologous PBMCs stimulated with anti-CD3/CD28 Dynabeads (1 × 10 5 beads/ml, ThermoFisher) in RPMI 1640 media supplemented with 10% heat inactivated FBS and 1% penicillin/streptavidin. At day 5 post stimulation, cells were stained with BD Horizon Fixable Viability stain 780, anti-CD3 PerCP Cy5.5, and anti-CD25 PE (BD Biosciences). Samples were analyzed by ow cytometry using BD FACSCelesta. Tregs were excluded based on their CD25 hi expression and lack of CellTrace Violet stain. T cell proliferation was assessed by measuring CellTrace Violet stain dilution within the CD3 + T cell population.
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