For analysis of PD-1, cells were washed in serum-free PBS and stained with a fixable viability dye, eFluor506, CD4-PerCPCy5.5, PD1-PECY7, CD8-APCeFluor780 (eBioscience), incubated for 15 min at room temperature in the dark, and then washed in PBS buffer containing 1% FBS and sodium azide. Cells were stained for Treg cell markers using a FoxP3 staining buffer set (eBioscience) and accompanying protocol. Treg cell markers included CD39-FITC, FoxP3-PE, CD73-PerCPeFluor710, CD25-PECY7, CTLA-4-APC, CD127-APCeFluor780, Ki67-eFluor450 (eBioscience), and CD4-V500 (BD Biosciences). An intracellular staining kit (Fix and Perm kit, Invitrogen) was used to analyze cytokine production after restimulation with PMA/ionomycin. Cells were stained with IL-17A-AlexaFluor488, IL-10-PE, TNF-α-PerCPCy5.5, CD45RA-PECY7, CD8-APCeFluor780, FoxP3-eFluor450 (all eBioSciences), CD45 AlexaFluor700 (BioLegend), IFN-γ-APC, CD3-V500, and IL-2-PE-CF594 (BD Biosciences). Due to PMA/ionomycin-mediated reduction in CD4 expression, CD4+ T cells were identified as CD3+CD8− T cells for cytokine analysis. Cells were acquired on a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Flowjo LLC).
Il 10 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
IL-10-PE is a fluorescent-labeled antibody product used for the detection and quantification of the cytokine interleukin-10 (IL-10) in biological samples. It is a tool for research purposes and its core function is to serve as a reagent for flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe, by Thermo Fisher Scientific (5 mentions)
Cd4 fitc, by Thermo Fisher Scientific (5 mentions)
Cd25 pe cy7, by Thermo Fisher Scientific (3 mentions)
Cd25 pe, by Thermo Fisher Scientific (3 mentions)
Cd86 pe, by Thermo Fisher Scientific (3 mentions)
Foxp3 pe, by Thermo Fisher Scientific (3 mentions)
Cd4 percp cy5, by Thermo Fisher Scientific (2 mentions)
Ctla 4 apc, by Thermo Fisher Scientific (2 mentions)
Foxp3 apc, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions)
Cd11b apc, by Thermo Fisher Scientific (2 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Foxp3 pe, by BioLegend (2 mentions)
Cd25 apc, by BioLegend (2 mentions)
Golgistop, by BD (2 mentions)
Ifn γ apc, by Thermo Fisher Scientific (2 mentions)
Il 17 pe, by Thermo Fisher Scientific (2 mentions)
Ifn γ pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd11c apc, by Thermo Fisher Scientific (2 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
20 protocols using il 10 pe
1
Multiparametric Flow Cytometry for Immune Profiling
2
Characterization of Adrenergic Receptor Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies: anti-alpha 1 adrenergic receptor (α1-ADR, Abcam, ab3462, Cambridge, UK); anti-beta 2 adrenergic receptor (β2-ADR, Abcam, ab36956); anti-beta-Arrestin-2 (β-Arrestin-2, Cell Signaling, Cambridge, UK, Clone: C16D9); anti-cyclic AMP-response element binding protein (CREB, Abcam, Clone: E306); anti-phospho-cyclic AMP-response element binding protein (pCREB, Abcam, Clone:E113); anti-extracellular regulated kinase 1/2 (ERK1/2, ThermoFisher Scientific, Waltham, MA, USA, Clone: K.913.4); anti-phospho-extracellular regulated kinase 1/2 (pERK1/2, Cell signaling, Clone: D13.14.4E; anti-G-protein-receptor-kinase-2 (GRK-2, Abcam, Clone: Y137); Interleukin-10-Phycoerythrin-conjugated (IL-10-PE, eBioscience, Frankfurt, Germany, Clone: JES5-16E3); anti-p38 mitogen activated kinase (p38 MAPK, Cell signaling, Clone: D13E1); anti-phospho-p38 mitogen activated kinase (pp38 MAPK, Cell Signaling, Clone: D3F9).
Secondary antibodies: goat anti-rabbit IgG biotin (Dako, Frankfurt, Germany; catalog number: E0432); goat anti-rabbit IgG-R-PE (Sigma-Aldrich, St. Louis, MI, USA, catalog number: P9537); Streptavidin-PE (eBioscience, ThermoFisher Scientific, catalog number: 12-4317-87).
Isotype controls: mouse IgG (Abcam, ab37355); rabbit IgG (Abcam, ab172730, Clone: EPR25A).
Secondary antibodies: goat anti-rabbit IgG biotin (Dako, Frankfurt, Germany; catalog number: E0432); goat anti-rabbit IgG-R-PE (Sigma-Aldrich, St. Louis, MI, USA, catalog number: P9537); Streptavidin-PE (eBioscience, ThermoFisher Scientific, catalog number: 12-4317-87).
Isotype controls: mouse IgG (Abcam, ab37355); rabbit IgG (Abcam, ab172730, Clone: EPR25A).
3
Quantification of Immune Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immune infiltration into adipose and liver tissues and splenic composition was quantified by flow cytometry as previously described13 (link),22 (link)–25 (link). Briefly, single cell suspensions from indicated tissues were obtained by enzymatic digestion. To determine cytokine production, total single cells were stimulated for 5 h with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 1 μg/ml Ionomycin (Calbiochem), in presence of brefeldin A (10 μg/mL, Sigma-Aldrich). Subsequently, flow cytometry was used to enumerate immune cell populations. Briefly, cells were incubated in PBS supplemented with 2% FBS and were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly conjugated monoclonal antibodies to CD3-AF700(145-2C11), TCRβ-BV711 (H57-597), CD8-PECʏ−7 (53-6.7), CD4-APC (RM4-5) (all antibodies from eBioscience) for 30 min. For intracellular staining, cells were fixed and permeabilized using eBioscience buffer and stained with FoxP3-PB (FJK-16s) and IL-10-PE (JES5-16E3). Flow cytometry data were collected using an LSR Fortessa (BD) flow cytometer and analyzed using FlowJo X software (vX0.7) and FACS Diva Software.
4
Multiparameter Analysis of Memory T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Memory CD4+ T cells were stimulated with phorbol 12-myristate acetate (PMA) (50 ng/mL) and ionomycin (1 µg/mL) for about 10 hours, with the last 4 hours with 10 µg/mL Brefeldin A and 2 µM monensin at 37°C and 5% CO2. In some instances, cells were first treated with CSA (1 µM) for 1 hour before PMA/Ionomycin treatment. After 10 hours, cells were fixed and permeabilized with the Foxp3 staining kit (eBioscience, San Diego, CA), according to the manufacturer's protocol. The following antibodies were used: interferon-γ (IFN-γ) FITC, interleukin (IL) 17–Alexa Fluor 647, tumor necrosis factor α (TNFα)–Alexa Fluor700, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) PECF594, IL-2 BV510, IL-10 PE, Foxp3 APC, RORγc PE, and PD1 BV605 (all eBioscience). Stained cells were acquired by CytoFLEX S (Beckman Coulter) and analyzed with the CytExpert Software.
5
Cytokine Profiling of Activated PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pooled human PBMCs were purchased from Zenbio and recovered overnight before stimulation. LPS (100 ng/mL) and 25 uL human CD3/CD28 activator solution / million cells and the treatments were added for 72 hours. Then PBMCs were cultured with 1 x Cell Stimulation Cocktail plus protein transport inhibitors (Invitrogen) for 4 hs. Single cells were harvested and stained with anti-human CD3-APCcy7 and CD14-FITC for 15 mins at room temperature. After that, the cells were fixed and permeabilized with the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Bioscience) and labeled intracellularly with anti-human IFNγ-APC, TNFα-BV605 (Biolegend) and IL10-PE (ebioscience). Data were collected on Attune NxT Flow Cytometer (Thermofisher) and analyzed using FlowJo software.
6
Isolation and Characterization of Liver Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver cells were obtained based on methods described by Nemeth et al Briefly, freshly removed liver tissues were minced into small pieces and incubated in RPMI 1640 medium with 300 U/mL collagenase type I (Sigma), 300 U/mL collagenase type IV (Sigma) and 50 U/mL DNase I (Sigma). After incubation, cell suspensions were filtered through a 70-um cell strainer and then washed with complete RPMI medium. Single-cell suspensions were incubated with an Fc receptor blocker (CD16/32, eBioscience) to reduce nonspecific antibody binding. The panel of antibodies used in these experiments included CD11b-APC, F4/80-FITC, IL-10 PE (all from eBioscience), CD4-FITC, CD25-APC and Foxp3-PE (all from Biolegend). Among these antibodies, CD11b, F4/80, CD4 and CD25 cause cell surface staining, while IL-10 and foxp3 result in both cell surface and intracellular staining. Flow cytometry was performed using a FACS Calibur flow cytometer (BD Bioscience), and data were analyzed using FlowJo software (TreeStar, Ashland, OR).
7
Multicolor Flow Cytometry for Immune Cell Profiling
8
Flow Cytometric Characterization of BMDCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The BMDCs were incubated with mouse Fc block (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°. Fluorochrome‐labelled monoclonal antibodies used in the experiments included anti‐mouse CD80‐allophycocyanin (APC), CD86‐phycoerythrin (PE), MHC‐II‐PE, CD11c‐APC, CD40‐APC, CD83‐PE, CCR7‐PE, CCR5‐APC, CXCR3‐APC, programmed death‐ligand 1 (PD‐L1) ‐PE, CD3e‐FITC, CD4‐PE, CD8‐APC, IL‐17‐PE, IL‐10‐PE, interferon‐γ (IFN‐γ) ‐PE‐Cy7, Foxp3‐APC and CD25‐PE (eBioscience) and their corresponding isotypic controls. For intracellular cytokine staining, cells were pre‐treated with Golgi Stop (BD Biosciences, Franklin Lakes, NJ) and were then stained with fluorochrome‐labelled surface staining monoclonal antibodies or isotypic control for 30 min. A Cytofix Cytoperm kit (BD Biosciences) was then used to permeabilize and fix cells at 4° for another 30 min. Perm wash buffer was used to wash the processed cells before staining them with anti‐IFN‐γ, anti‐IL‐17 and anti‐IL‐10 monoclonal antibodies and isotypic controls for 30 min at 4°. Anti‐mouse Foxp3 staining set APC (eBioscience) was used for Foxp3 immunostaining. Data were captured by flow cytometry (Beckman Coulter, Brea, CA) and analysed using the flowjo software (FlowJo, Ashland, OR).
9
Comprehensive T-cell Phenotyping by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspension (106 cells/tube) was incubated with anti-CD16/32 antibody to block Fc receptors for 15min at 4°C. For surface staining, the cells were stained with antibodies or matched isotype control for 20 min at 4°C in the dark. The antibodies included anti-mouse CD3-PECY7, CD4-FITC, CD8-Percpcy5.5, CD25-APC, CD11c-PE, CD86-APC, CD80-PerCP-eFlour710, PD-L1-PECY7, MHCII-Alexa Flour 700, MHCII- APC, and CD103-FITC antibodies (all from Biolegend, San Diego, CA, USA). For intracellular staining, cells were cultured in the presence of 2μl/ml cell stimulation cocktail (eBioscience) for 6 h. The cells were fixed and permeabilized using fixation and permeabilization solutions, then stained with anti-mouse Foxp3-PE, IL4-PE, IFN-γ-APC, IL10-PE antibodies (all from eBioscience), or isotype control for 30 min at 4°C in the dark. The cells were detected using BD FACSVerse (BD Biosciences, Franklin Lakes, NJ, USA) or Aurora (Cytek, Fremont, CA, USA), and data were analyzed using the FlowJo 10.0.7 software (Tree Star, Ashland, OR, USA).
10
Comprehensive Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for flow cytometry analysis: CD11c APC, CD80 FITC, CD83 FITC, CD86 PE, HLA-DR FITC, CD1a FITC, CD14 FITC, CD4PE, CD4 PE-Cy7, CD45RA PE, CD45RO FITC, CD25 PE-Cy7, CTLA-4 APC, CD39 PerCP-eFluor710, FOXP3 Alexa Fluor 700, IL-10 PE, IFN-γ PE-Cy7, and IL-17 APC (all eBioscience). For surface staining, cells were incubated in PBS 10% FBS containing the respective antibodies for 30 min at 4°C, washed, and stored in IC fixation buffer until analysis (eBioscience). Intracellular FoxP3 was detected using FoxP3-staining kit (eBioscience) according to the manufacturer’s instructions. For intracellular cytokines detection, cells were treated with 50 ng/ml PMA, 1 μg/ml ionomycin, and 1 μl/ml brefeldin A for 5 h. After harvesting, cells were surface stained for CD4. Intracellular cytokine staining was performed in permeabilization buffer (eBioscience) before cells were washed and resuspended in FACS buffer for flow cytometry analysis. Cell viability was assessed by 7-AAD and annexin-V PE staining (eBioscience). Data were collected on FACSCalibur and FACSAria cytometers (Beckton Dickinson, San Diego, CA, USA) and analyzed with Weasel v3.0.2 software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!