Pieces of pancreas were removed, weighed, and placed in a tube containing 700 μl Complete Protease Inhibitor Cocktail (Roche Diagnostics). Pancreatic tissue was homogenized using a Polytron homogenizer (Kinematica, Luzern, Switzerland) and IL-2, IL-6, IFNγ, TNFα, IL-17, IL-4, and IL-10 levels were detected by the cytometric bead array (CBA) (Th1/Th2/Th17 kit; BD) method, according to the manufacturer’s instructions. The concentration of TGF-β in pancreatic tissue was determined using Human/Mouse TGF-β1 ELISA Ready-Set-Go kit (eBioscience, San Diego, CA, USA). Serum cytokine levels were also determined by the CBA method.
Th1 th2 th17 kit
Manufactured by BD
Sourced in United States, Switzerland
The Th1/Th2/Th17 kit is a laboratory tool used for the detection and quantification of T helper cell subsets, which are important for understanding immune responses. The kit provides the necessary reagents and protocols to measure the levels of Th1, Th2, and Th17 cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (4 mentions)
Human mouse tgf β1 elisa ready set go kit, by Thermo Fisher Scientific (2 mentions)
Polytron homogenizer, by Kinematica (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Ccr7 pe cy7, by BD (2 mentions)
Cd45 v500, by BD (2 mentions)
Cd4 pe cy7, by Thermo Fisher Scientific (2 mentions)
Ccr5 pe, by BD (2 mentions)
Cd4 apc h7, by BD (2 mentions)
Cd45ro pe, by Thermo Fisher Scientific (2 mentions)
Cxcr3 alexa fluor488, by BD (2 mentions)
Ccr4 percp cy5, by BD (2 mentions)
Ccr6 alexa647, by BD (2 mentions)
Cd69 percp, by BD (2 mentions)
Cd45ra efluor450, by Thermo Fisher Scientific (2 mentions)
Cd3 v500, by BD (2 mentions)
Facscalibur, by BD (1 mentions)
Cd8 v500, by BD (1 mentions)
Cd8 apc efluor 780, by Thermo Fisher Scientific (1 mentions)
Foxp3 percp cy5, by BD (1 mentions)
12 protocols using th1 th2 th17 kit
1
Cytokine Profiling in Pancreatic Tissue
2
Evaluating NKG2D CAR-T Cells in NSG Mice
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Five-to-6-week-old female NOD SCID gamma (NSG) mice (GemPharmatech) were intraperitoneally injected with 5×106 A549-Luc-green fluorescent protein (GFP) cells. These cancer cells in the mice were then detected by live bioluminescent imaging. Images were collected and analyzed with a Xenogen-IVIS Imaging System. When the mean fluorescence intensity (MFI) value was >109, the mice were divided into three groups, which received NKG2D(z) CAR-T cells (8×106/mouse), NKG2D(bbz) CAR-T cells (8×106/mouse), or mock-T cells (ethics ID number: Xmsq2021-0075). The weight of the mice and MFI values were regularly monitored. The supernatant was aspirated after centrifugation of peripheral blood from the mice (collected via the submandibular vein) and the secretion of IL6, IL2, IFN-γ, and TNF-α in the peripheral blood was measured with a Th1/Th2/Th17 kit (560484, BD Pharmingen). The percentages of NKG2D(z) and NKG2D(bbz) CAR-T cells were assessed based on mCherry fluorescence after lysis of erythrocytes.
3
Cytokine Profiling in Ischemic Stroke
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IL-6, CCL2, TNF-α, IFN-γ, IL-10 and IL-12p70 proteins contained in protein lysates from the contralateral and ipsilateral cortices of sham-operated and ischemic WT and ColXV KO mice treated or not with tPA were measured by using the cytometric bead assay Th1/Th2/Th17 kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Data were acquired using FACSCalibur (BD Biosciences, San Jose, CA, USA) and analyzed by FCAP Array software (Soft Flow, St. Louis Park, MN, USA).
4
Flow Cytometry Panel for Immune Cell Profiling
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Cells were stained for 30 min at 4 °C in PBS containing 0.01% NaN3 and 0.5% BSA with directly labelled antibodies against: CXCR3 alexa fluor488, CCR5 PE, CCR4 PercP-Cy5.5, CCR7 PE-Cy7, CCR6 alexa647, CD4 APC-H7,CD8 V500, CD3 V500, CD69 PercP, CD45 V500, Foxp3 PercP-Cy5.5, Granzyme-A PE, CD8 V450 (all from BD Biosciences), CD3 FITC (Sanquin, Amsterdam, The Netherlands), CD4 PE-Cy7, CD28 APC, CD8 APC-efluor780, CD45RA efluor450, CD45RO PE (all from eBioscience Inc., San Diego, CA, USA), Granzyme-K Fitc (Immunotools, Friesoythe, Germany), Granzyme-B APC (Invitrogen, Thermo Fisher Scientific Inc.) and Perforin PercP-Cy5.5, IL-10 Pe-Cy7 (Biolegend, San Diego, CA, USA). For cytokine staining, we used the Th1/Th2/Th17 kit from BD Biosciences. Cells were analysed on an FACS Canto II (BD Biosciences) and data were analysed using the FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI to illustrate cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest was normalized to the gMFI of the negative population.
Data were plotted as frequency of positive cells or as the gMFI to illustrate cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest was normalized to the gMFI of the negative population.
5
Cytokine and NO Quantification Protocol
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Cytokine levels in the culture supernatants were measured by cytometric bead array (Th1Th2Th17 kit from BD Biosciences and Th1Th2Th17Th22 13-plex from eBioscience, San Diego, CA USA) or ELISA. All ELISA reagents were purchased from BD Biosciences with the exception of rIL-17 (eBioscience) and used according to manufactures instructions. NO production was measured in culture supernatants by Griess reaction as described [11 (link)].
6
Cytokine Profiling in Arthritis
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The levels of different cytokines (IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-gamma and IL-17) were assessed in plasma and synovial fluid samples by cytometric bead array (CBA) with a Th1/Th2/Th17 kit (BD Biosciences, Sao Paulo, Brazil); according with the manufacturer’s instructions. Data collection and analysis were performed in a BD Accuri C6 flow cytometer. Results were calculated in CBA FCAP Array software (BD Biosciences, Sao Paulo, Brazil) and are expressed as picograms of cytokine per millilitre (pg/mL).
7
Multiparametric Flow Cytometry Immunophenotyping
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Cells were stained for 30 min at 4°C in PBS containing 0.01% NaN3 and 0.5% BSA with directly labeled antibodies against: CXCR3 Alexa fluor488, CCR5 PE, CCR4 PerCP‐Cy5.5, CCR7 PE‐Cy7, CCR6 Alexa647, CD4 APC‐H7, CD3 V500, CD69 PerCP, CD45 V500 (all from BD Biosciences); CD3 FITC (Sanquin); CD4 PE‐Cy7, CD45 RA eFluor450, and CD45RO PE (all from eBioscience Inc., San Diego, CA). For intracellular cytokine staining, we used IL‐10 PE‐Cy7 (BioLegend, San Diego, CA, USA) and the Th1/Th2/Th17 kit from BD Biosciences. For intracellular staining, after cell surface staining, cells were washed and fixed with FACS Cytofix fixation buffer (BD Biosciences). Following permeabilization (FACS Perm/Wash buffer [BD Biosciences]) cells were stained with markers as indicated. Cells were analyzed on a FACS Canto II (BD Biosciences). CS&T beads were run daily and the same machine with dedicated cytometer configuration was used for the measurements of the samples throughout the study with regular control runs to check compensation settings. Data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI for cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest were normalized to the gMFI of the cytokine negative population.
Data were plotted as frequency of positive cells or as the gMFI for cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest were normalized to the gMFI of the cytokine negative population.
8
Cytokine Profiling in Spleen Cells
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Cytokine levels of TNF, MCP-1, IL-10, IL-6, IL-2, IFN-γ, IL-17A, and IL-4 in the supernatants from cultured spleen cells and macrophage were measured using the cytometric bead array (CBA) mouse inflammation kit or Th1/Th2/Th17 kit (BD Biosciences, CA, United States), according to the recommendations of the manufacturer. Briefly, the samples were incubated for 3 h at room temperature with a mixture of fluorescent capture spheres covered with specific antibody for each cytokine and fluorescently labeled detection antibodies. After this time, the samples were washed with the solution provided by the manufacturer. Around 2,500 events were acquired in FACS Canto II flow cytometer (Becton Dickinson, Mountain View, CA, United States) using the FACS Diva software. Data analysis was performed using the FCAP Array program (BD Biosciences) and the concentration of cytokines was determined based on the established standard curve, according to the guidelines of the manufacturer.
9
Cytokine Profiling of Activated Splenocytes
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Splenocytes (1×106) were incubated with PMA (50 ng/ml; Sigma)) and ionmycin (1 µg/ml; Sigma) in complete media (200 µl/well) containing Golgi stop (1∶1500 dilution; BD BioSciences). Following an incubation (5 h, 37°C, 5% CO2), cells were centrifuged (350 g, 5 min) and re-suspended in Fixable viability dye eFluor 450 (1∶1000 in PBS; eBioscience). Following 30 min incubation (4°C), cells were centrifuged (350 g, 5 min), supernatant aspirated, cells re-suspended in Cytofix (BD Biosciences), and incubated for a further 15 min (room temperature). Cells were washed (x2 stain buffer), before re-suspending in perm/wash buffer (BD Biosciences) and incubated for 15 min (room temperature). Cells were pelleted (350 g, 5 min) and re-suspended in 50 µl perm/wash buffer containing 20 µl staining cocktail (Th1/Th2/Th17 kit; BD Biosciences) or negative controls. Following a further incubation (30 min, room temperature), cells were washed (x2 stain buffer), re-suspended in PBS and analysed on BD FACS Canto II as above.
10
Multiplex Analysis of Pancreatic Cytokines
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Pieces of pancreas were removed, weighed and placed into a tube containing Complete Protease Inhibitor Cocktail (Roche Diagnostics, Abbott Park, IL, USA). Pancreatic tissue was homogenized using a Polytron homogenizer (Kinematica, Luzern, Switzerland) and interleukin (IL)-2, IL-6, interferon gamma, IL-17, IL-4 and IL-10 levels were detected in the supernatant by the cytometric bead array method (Th1/Th2/Th17 kit; BD), according to the manufacturer’s instructions. The concentration of transforming growth factor beta (TGF-β) in pancreatic tissue was determined using the Human/Mouse TGF-β1 ELISA Ready-Set-Go kit (eBioscience, San Diego, CA, USA). Serum cytokine levels were also determined by the cytometric bead array method.
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