Cells were stained with mouse CD11c (N418, 1/200, eBioscience), F4/80 (BM8, 1/200, Biolegend), or TCR-β (H57-597, 1/200, Biolegend) antibodies at 37°C for 10 min, stimulated with cytokines for 15 min, fixed with 2% paraformaldehyde at 37°C for 10 min, permeabilized in 90% ice-cold methanol for 30 min, washed twice with FACS buffer (PBS with 1% FBS), stained with pSTAT3 (Y705)-PE or pSTAT3 (Y705)-Alexa Fluor 647 antibodies (4/P-STAT3, 1/25, BD Biosciences) at RT for 30 min, and analyzed on a FACSCanto II (Becton Dickinson).
H57 597
Manufactured by BioLegend
The H57-597 is a monoclonal antibody produced by BioLegend that binds to the hamster CD3 epsilon chain. It is commonly used in flow cytometry and other immunoassays to detect and analyze T cells.
Automatically generated - may contain errors
Lab products found in correlation
Fjk 16, by Thermo Fisher Scientific (4 mentions)
M5 114, by BioLegend (3 mentions)
Facscanto 2, by BD (2 mentions)
4 p stat3, by BD (2 mentions)
M1 70, by BioLegend (2 mentions)
Anti cd28 mab, by BioLegend (2 mentions)
Facsaria 2, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
M1 69, by BioLegend (2 mentions)
Michel 19, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Fortessa, by BD (2 mentions)
Collagenase 4, by Merck Group (2 mentions)
Cd4 gk1, by BioLegend (2 mentions)
Sa011f11, by BioLegend (2 mentions)
Cd25 pc61, by BioLegend (2 mentions)
Apc efluor 780, by Thermo Fisher Scientific (1 mentions)
19 protocols using h57 597
1
Phosphorylated STAT3 Quantification
2
Multiparameter Flow Cytometry Assay
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Antibodies against murine CD4 (RM4-5, AF700, BV650), CD8 (53-6.7, APC/Fire 750, BV785), CCR2 (SA203G11, BV421), CD11c (N418, APCCy7, Pe/Cy5), CD206 (C068C2, APC), CD11b (M1/70, BV421), F4/80 (BM8, BV510, BV711, FITC), TCRβ (H57-597, BV605), and NK1.1 (PK136, BV650) were purchased from BioLegend. Antibodies against CD4 (GK1.5, BUV395) and ST2 (U29-93, BV480) were purchased from BD Biosciences. Antibodies against B220 (RA3-6B2, AF488), CD11c (N418, e450, PE-Cy5.5), CD25 (PC61.5, PE-Cy7), CD4 (GK1.5, APC-eFluor 780, Super Bright 645), CD45 (30-F11, Pacific Orange), Foxp3 (FJK-16s, APC, e450, FITC), GITR (DTA-1, Super Bright 600), IL-10 (JES5-16E3, PE), KLRG1 (2F1, APC, APC-eFluor 780, PE-eFluor 610), NK1.1 (PK136, PE), and ST2 (RMST2-2, PE, PerCP-eFluor710) were purchased from eBioscience, Thermo Fisher Scientific. Viability dyes (e506, near IR, UV) were purchased from Invitrogen, Thermo Fisher Scientific.
Extracellular flow staining was performed in 2% FBS-PBS in the presence of BioLegend’s TruStain FcX for 30 minutes at 4°C. Fixation, permeabilization, and intracellular staining were performed using eBioscience’s (Thermo Fisher Scientific) Foxp3/transcription factor staining buffer set per manufacturer’s directions. Flow data were collected using a Cytek Aurora or BD Biosciences LSR and analyzed using FlowJo.
Extracellular flow staining was performed in 2% FBS-PBS in the presence of BioLegend’s TruStain FcX for 30 minutes at 4°C. Fixation, permeabilization, and intracellular staining were performed using eBioscience’s (Thermo Fisher Scientific) Foxp3/transcription factor staining buffer set per manufacturer’s directions. Flow data were collected using a Cytek Aurora or BD Biosciences LSR and analyzed using FlowJo.
3
Comprehensive Immune Cell Profiling
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Cells harvested after overnight incubation were stained following standard protocols. Experiments used antibodies specific for human FcR block (BioLegend #422302), CD11c (Bu15, BioLegend #337214), CD80 (2D10, BioLegend #305220), CD86 (Bu63, BioLegend #374210) HLA-DR (L243, BioLegend #307629), HLA-A,B,C (W6/32, BioLegend #311410), CD14 (M5E2, BioLegend #301828), CD63 (H5C6, BioLegend #353007), CD61 (VI-PL2, BioLegend #336416), CD41/CD61 (PAC-1, BioLegend 362804), CD62p (AK4, BioLegend #304906), mTCRb (H57-597, BioLegend #109206), CD4 (OKT4, BioLegend #317428), CD8 (SK1, BioLegend #344704), and human CD3-APC (Clone OKT3, BioLegend). Color-matched isotype control antibodies were obtained from the same vendors. Flow cytometry was performed with a Stratedigm flow cytometer with electronic gates set on live cells by a combination of forward and side light scatter and 7-AAD (BioLegend), EMA (Invitrogen), Zombie Aqua (BioLegend), or Zombie NIR (BioLegend) exclusion. A minimum of 3 × 104 events were collected per sample, and data were analyzed with FlowJo software (FlowJo LLC).
4
Naive CD4 T Cell Activation and Differentiation
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Naive CD4 T (CD44lowCD62LhighCD25negative) cells were prepared using a CD4+CD62L+ T-cell isolation kit II (Miltenyi Biotec). Naive CD4 T cells (1.5 × 106) were stimulated with immobilized anti-TCR-β mAb (3 μg ml−1, H57-597, BioLegend) and an anti-CD28 mAb (1 μg ml−1, 37.5, BioLegend) for 2 days under the conditions indicated. Next, cells were transferred to a new plate and cultured further in the presence of cytokines. Cytokine conditions were as follows: IL-2 (TH) conditions: IL-2 (2.5 ng ml−1); neutral conditions: IL-2 (2.5 ng ml−1), anti-IL-4 mAb (5 μg ml−1, 11B11, BioLegend) and anti-IFN-γ mAb (5 μg ml−1, R4-6A2, BioLegend).
5
Isolation and Activation of Naïve and Total CD8+ T Cells
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CD8+ T cells were prepared from spleen using a MojoSort Mouse CD8+ T cell isolation kit (cat#480035; BioLegend). In naïve CD8+ T (CD44lowCD62Lhigh) cell preparation, biotin anti‐CD44 mAb (cat#103004; BioLegend) was added. In the case of total CD8+ T cells, anti‐CD44 mAb was not added. After isolation, the cells (7.5 × 105) were stimulated with immobilized anti‐TCR‐β mAb (3 μg/mL, H57‐597; BioLegend) and anti‐CD28 mAb (1 μg/mL, 37.5; BioLegend) with IL‐2 (10 ng/mL; cat#575406; BioLegend) for 2 days. The cells were then transferred to a new plate and further cultured with IL‐2 (10 ng/mL) for 5 days.
6
Phosphorylated STAT3 Expression Assay
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Cells were stained with mouse CD11c (N418, 1/200 and eBioscience), F4/80 (BM8, 1/200, Biolegend), or TCR-β (H57-597, 1/200, Biolegend) antibodies at 37 °C for 10 min, stimulated with cytokines for 15 min, fixed with 2% paraformaldehyde at 37 °C for 10 min, permeabilized in 90% ice-cold methanol for 30 min, washed twice with FACS buffer (PBS with 1% FBS), stained with pSTAT3 (Y705)-PE or pSTAT3 (Y705)-Alexa Fluor 647 antibodies (4/P-STAT3, 1/25 and BD Biosciences) at RT for 30 min, and analysed on a FACSCanto II (Becton Dickinson).
7
Multiparametric Flow Cytometry Panel
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The following clones of antibody were purchased from Biolegend and used for cell surface staining: CD4 (RM4-5), CD8 (53-6.7), TCRβ (H57-597), B220 (RA3-6B2), CD69 (H1.2F3), CD25 (PC61), Vβ5 (MR9-4), IgM (RMM-1), IgD (11-26c.2a), CD21 (7E9), CD23 (B3B4). Flow cytometry was performed on a FACSCanto or FACSCanto II and analyzed with FlowJo software.
8
Multiparameter Flow Cytometry Panel
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PD-1 FITC (1:300; J43; eBioscience), NK1.1 PE (1:300; PK136; eBioscience), CD3e PE-Dazzle594 (1:300; 145-2C11; BioLegend), CD3e PE (1:200; 145-2C11; eBioscience), FoxP3 PerCP-Cy5.5 (1:200; FJK-16s; eBioscience), CD8a PE-Cy7 (1:2000; 53-6.7; eBioscience), CD8a APC (1:600; 53-6.7; eBioscience), CD8a PerCP-Cy5.5 (1:400; 53-6.7; eBioscience), TCRb AF700 (1:100; H57-597; BioLegend), CD45 BV785 (1:1000; 30-F11; BioLegend), CD4 BV711 (1:400; GK1.5; BioLegend), CD19 BV650 (1:400; 6D5; BioLegend), CD19 PE (1:500; 1D3; eBioscience), CD11b BV605 (1:1000; M1/70; BioLegend), CD11b BV510 (1:400; M1/70; BioLegend), CD11c BV605 (1:400; N418; BioLegend), Siglec-F PE (1:300; E50-2440; BD Biosciences), F4/80 APC (1:200; BM8; BioLegend), Ly-6G AF 700 (1:300; 1A8; BioLegend), MHC II BV650 (1:4000; M5/114.15.2; BioLegend), CD64 BV421 (1:200; X54-5/7.1; BioLegend), TNF FITC (1:300; MP6-XT22; BioLegend), IFNg PE-Cy7 (1: 1000; XMG1.2; BD Biosciences), Eomes PE (1:200; W17001A; BioLegend), Ki-67 BV 650 (1:200, 11F6; Biolegend), GzmB FITC (1:100; GB11; BioLegend). Gating strategy is depicted in Supplementary Fig. 6d .
9
Comprehensive Thymocyte and TEC Profiling
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Thymocytes were incubated with Abs against TCR beta (1:400, H57-597; Biolegend), CD4 (1:1000, GK1.5; BioLegend), CD5 (1:400, 53-7.3, eBioscience), CD8 (1:500, 53-67; BioLegend), CD25 (1:1000, PC61.5; BioLegend), CD44 (1:500, IM7; BioLegend), c-kit (1:200, 2B8; BioLegend), CD24 (1:1000, M1/69; BioLegend), CD69 (1:200, H1.2F3; BioLegend), PD-1 (1:200, 29F.1A12, Biolegend), CCR7 (1:200, 4B12, BioLegend). For intracellular staining, cells were fixed, permeabilized (Fixation/Permeabilization kit, eBioscience) and stained for Foxp3 (1:70, FJK-16S, eBioscience) and Helios (1:70, 22F6, eBioscience). TECs were stained using Abs against CD45 (1:400, 30F11; BioLegend), EpCAM (1:1000, G8.8; BioLegend), MHCII (1:1000, M5/114.15.2; BioLegend), Ly51 (1:1000, 6C3; BioLegend), UEA-1 (1:1000, Reactolab), CD80 (1:500, 16-10A1, Biolegend), CD86 (1:500, GL-1, Biolegend), CD83 (1:500, Michel 19, Biolegend). For intracellular staining, cells were fixed, permeabilized (Cytofix/Cytoperm Kit, BD Biosciences) and labelled for the expression of CD83. Stained samples were acquired on a FACSAria II or BD LSRFortessa flow cytometers and the data was analyzed using the FlowJo (Treestar) software.
10
Naive CD8 T Cell Activation and Glutamine Deprivation
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Naive CD8 T (CD44lowCD62Lhigh) cells were prepared using a Naive CD8+ T-cell Isolation kit (cat#130-096-543; Miltenyi Biotec, San Diego, CA, USA). Naive CD8 T cells (1.5 × 106) were stimulated with immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) and anti-CD28 mAb (1 μg/ml, 37.5; BioLegend) for 2 days in the presence of IL-2 (10 ng/ml, Pepro Tech). The cells were then transferred to a new plate and further cultured in the presence of IL-2 (10 ng/ml). The cells were cultured in RPMI 1640 with l -glutamine (cat#189-02025; Wako Chemicals) supplemented with 10% fetal bovine serum, 2 mM l -glutamine (16948-04; Nacalai Tesque, Kyoto, Japan), 1 mM sodium pyruvate (cat#06977-34; Nacalai Tesque), 1% MEM nonessential amino acids (cat#06344-56; Nacalai Tesque), 10 mM HEPES (cat#15630-080; Thermo Fisher Scientific, Waktham, MA, USA), 55 μM 2-Mercaptoethanol (cat#21985-023; Thermo Fisher Scientific), and 1% penicillin-streptomycin (cat#26253-84; Nacalai Tesque). For glutamine-deprived conditions, the cells were cultured in RPMI 1640 without l -glutamine (cat#183-02165; Wako Chemicals) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% MEM nonessential amino acids, 10 mM HEPES, 55 μM 2-Mercaptoethanol, and 1% penicillin-streptomycin.
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