To analyze the expression of cytokine of CD4+ T cells, cell surface staining was performed using pacific blue-conjugated CD4 (Biolegend) for 20 min at 4 °C in the dark. After the stained cells were washed with PBS, intracellular staining was performed to detect TNF-α, IFN-γ, IL-17A, and IL-4 expression. According to the manufacturer’s instructions, the cells were fixed and permeabilized using a Transcription Factor Staining Buffer Set kit (eBiosciences) and stained with allophycocyanin (APC)-cy7 conjugated TNF-α, (phycoerythrin) PE-cy7-conjugated IFN-γ, IL-4, or APC-conjugated IL-17A (eBiosciences). Prior to cytokine analysis, the purified CD4+ T cells were restimulated with 40 ng/mL phorbol-12-myristate-13-acetate (Sigma-Aldrich) and 1 µg/mL ionomycin (Sigma-Aldrich) for 5 h. Monensin (Sigma-Aldrich) was added at a concentration of 4 µM for the last 1 h of TCR stimulation or the last 6 h of PHA stimulation.
Transcription factor staining buffer set kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Transcription Factor Staining Buffer Set kit is a laboratory tool designed for the detection and analysis of transcription factors in cell samples. The kit provides the necessary buffers and reagents to facilitate the staining and permeabilization of cells, allowing for the intracellular detection of transcription factors using flow cytometry or other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Monensin, by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Apc conjugated il 17a, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Fcr blocker, by BD (1 mentions)
Apc conjugated cd25, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Enzo Life Sciences (1 mentions)
Brefeldin a bfa, by Enzo Life Sciences (1 mentions)
Flow cytometry, by BD (1 mentions)
4 protocols using transcription factor staining buffer set kit
1
Cytokine Expression Profile of CD4+ T Cells
2
Multi-parameter Flow Cytometry Analysis
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Cells were blocked with FcR-blocker (BD PharMingen), and then incubated with monoclonal Abs for surface antigens. Fluorochrome-conjugated monoclonal Abs used in this study were: anti-mouse F4/80, CD205, anti-human CD68, CD205, CD14, CD16, HLA-DR, TNF-α, IL-12/23p40 (BioLegend), anti-mouse CD45, CD11b, Ly6C, CD14, CD40, CD80, CD86 (BD PharMingen). Intracellular staining was performed using the Transcription Factor Staining Buffer Set Kit (eBioscience) and Abs to human CD68, TNF-α, IL-12/23p40. All samples were analysed using FACSVerse flow cytometry and FlowJo 7.6.1 software.
3
Phenotypic Analysis of Induced Tregs
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Flow cytometric analysis was conducted to analyze the phenotypes of the induced Tregs. Cell surface staining was performed with fluorescein isothiocyanate (FITC)- or allophycocyanin (APC)-conjugated CD25 (eBiosciences), APC- or PE-conjugated ICOS (eBiosciences), or FITC-conjugated CD4 (eBiosciences) or PE-Cy7-conjugated CD127 (eBiosciences) for 20 min at 4 °C in the dark. After washing, intracellular staining was performed to detect FoxP3 or IL-10 expression. According to the manufacturer’s instruction, The cells were fixed and permeabilized using Transcription Factor Staining Buffer Set kit (eBiosciences), and then stained with FITC- or PE-conjugated Foxp3 (eBiosciences) or PerCP-Cy5.5- or PE-conjugated IL-10 (eBiosciences). Prior to IL-10 analysis, the cells were stimulated with 40 ng/mL phorbol-12-myristate-13-acetate (Sigma-Aldrich) and 1 μg/mL ionomycin (Sigma-Aldrich) for 5 h. Monensin (Sigma-Aldrich) was added at a concentration of 4 μM for the last 1 h of stimulation.
4
Quantifying Treg and Th17 in MMD
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To investigate the changes of Treg and Th17 cells in MMD patients, flow cytometry was used to measure the percentage of Th17 and Treg cells. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood by Ficoll gradient centrifugation. For Th17 cells, the isolated PBMCs were pre-treated with phorbol 12-myristate-13-acetate (PMA, 25 ng/ml), ionomycin (50 µg/ml) and brefeldin-A(BFA, 10 µg/ml, Enzo Life Sciences, Farmingdale, USA) at 37 °C for 4 h, then were collected and surface-stained with antibodies (eBiosicence, Frankfurk, Germany) at room temperature (RT) for 15 min, followed by fixed and permeabilized with the transcription factor staining buffer set kit according to the manufacturer’s protocol (eBioscience), then intracellular-stained with IL-17 antibody (eBioscience) for 30 min. Treg and its subtypes were incubated with surface antibodies at RT for 15 min. After fixed and permeabilized, cells were stained with Foxp3 antibody (eBiosicence) at RT for 30 min. Isotype controls were used in parallel. CD4+CD25+Foxp3+ cells were considered as Treg cells while CD3+CD8−IL-17+ cells were defined as Th17 cells. Cells were measured using flow cytometry (BD bioscience, San Diego, CA, USA). Data were analyzed using Flowjo software (Tree Star, Ashland, OR).
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