CD4 (5 µl) and CD25 (5 µl) (Additional file 1 : Table 1) were added to fresh MNCs (100 µl) that were from collected peripheral blood by disruption of red blood cells and incubated for 30 min at 4 °C in the dark. Foxp3 was stained by Intracellular Fixation & Permeabilization Buffer Set Kit and Foxp3/Transcription Factor Staining Buffer Set Kit (eBioscience, San Diego, CA, US). Fixation/Permeabilization Concentrate: Fixation/Permeabilization Diluent (v/v: 1/3) was prepared and added to the washed cells for membrane rupture (incubation for 60 min at 4 °C in the dark). After cell suspension was washed (2000 rpm, 5 min) by 1 × permeabilization buffer, Foxp3-PE (4 µl, Additional file 1 : Table 1) was added and incubated for 60 min at 4 °C in the dark. Next, the cell suspension was washed by permeabilization buffer (2500 rpm, 7 min) twice and resuspended to 200 µl for detection by flow cytometry (FC500, Beckman Coulter, Inc., Brea, CA, US).
Intracellular fixation permeabilization buffer set kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Intracellular Fixation & Permeabilization Buffer Set Kit is a laboratory product designed for the fixation and permeabilization of cells prior to intracellular staining and flow cytometry analysis. The kit includes a fixation buffer and a permeabilization buffer, allowing researchers to effectively prepare samples for the detection and analysis of intracellular targets.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (2 mentions)
Cd4 rm4 5, by Thermo Fisher Scientific (2 mentions)
Cd25 pc61, by BD (2 mentions)
Cd8 53 6, by Thermo Fisher Scientific (2 mentions)
Cd62l mel 14, by BD (2 mentions)
Live dead fixable red, by Thermo Fisher Scientific (2 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)
Cd44 im7, by Thermo Fisher Scientific (2 mentions)
Fc500, by Beckman Coulter (1 mentions)
Foxp3 transcription factor staining buffer set kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Pe anti mouse cd8, by Thermo Fisher Scientific (1 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
Anti mouse cd3 apc, by Thermo Fisher Scientific (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
6 protocols using intracellular fixation permeabilization buffer set kit
1
Quantification of Regulatory T Cells
2
Multiparametric Flow Cytometry Analysis
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LIVE/DEAD Fixable Red or LIVE/DEAD Fixable Aqua (Invitrogen) staining was performed at 4°C or on ice for 15 min in PBS. Surface Ab staining was performed at 4°C or on ice for 20 min in PBS containing 2% FBS and 2 mM EDTA. For staining intracellular transcription factors, fixation and permeabilization was performed with the eBioscience Intracellular Fixation & Permeabilization Buffer Set Kit prior to intracellular staining for 1 h at 4°C or on ice. Abs for flow cytometry included CD4 (RM4-5; Invitrogen), CD8 (53-6.7; eBioscience), CD25 (PC61; BD Biosciences), CD44 (IM7; eBioscience), CD62L (MEL-14; BD Biosciences), and Foxp3 (FJK-16S; eBioscience). Cells were analyzed or sorted on LSR II, LSRFortessa, and FACSAria II flow cytometers (BD Biosciences) with BD FACSDiva software. Data analysis was performed using FlowJo v.10.2 (Tree Star). FACS data presented in Fig. 3 are gated on forward scatter and side scatter properties indicative of singlet lymphocytes and live CD4+CD8−(CD4SP) cells.
3
Splenic T Cell and TIL Immunophenotyping
4
Multiparametric Flow Cytometry Analysis
5
Stimulation and Intracellular Cytokine Staining
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Fresh spleen cells and draining lymph node cells were isolated and stimulated with phorbol 12-myristate 13-acetate (PMA, 0.05 μg/mL) and ionomycin (0.5 μg/mL) for 1 h followed with brefeldin A (5 μg/mL) for 4 h at 37°C in a humidified tissue culture incubator with 5% CO2 and 95% O2. Then cells were collected, fixed, and permeabilized according to the manufacturer’s protocol of Intracellular Fixation & Permeabilization Buffer Set Kit (85-88-8824-00, eBioscience) before being stained with targeted FACS antibodies, such as CD4, CD8a, CD11b, Ly-6C, Gr1, IL-10, IL-17A, TNF-α, IFN-γ, and IL-4, followed by analyses with BD LSRFortessa Cell Analyzer.
6
Flow Cytometry Analysis of T Cell Subsets and Cytokines
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The percentages of CD4+ and CD8+ T lymphocytes subsets and the production of cytokine in the splenocytes of immunized mice were determined using flow cytometry techniques as described previously (Zhang et al., 2015b (link); Wang S. et al., 2017 (link)). Briefly, splenocytes (2 × 106cells/ml) of vaccinated mice were stimulated with G10E peptides (10 μg/mL) for 72 h. Then the suspensions were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE (eBiosciences, USA) for 30 min at 4 °C in the dark.
Splenocytes (2 × 106 cells/ml) were also stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail (eBiosciences, USA) containing Phorbol myristate acetate (PMA, 20 ng/ml), Ionomycin (2 μg/ml), Brefeldin A (1 μg/ml), and Monensin (1 μg/ml) to inhibit the secretion of cytokine into the extracellular space. The cells were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit in accordance with the manufacturer's protocol (eBiosciences, USA) and then stained directly with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC), and anti-mouse IL-10 (PerCP-Cyanine5.5) (eBiosciences, USA) for 30 min at 4°C. All these cell population were analyzed by Cytoflex S Flow Cytometer (Beckman Coulter, USA) and the data were analyzed by CytExpert software.
Splenocytes (2 × 106 cells/ml) were also stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail (eBiosciences, USA) containing Phorbol myristate acetate (PMA, 20 ng/ml), Ionomycin (2 μg/ml), Brefeldin A (1 μg/ml), and Monensin (1 μg/ml) to inhibit the secretion of cytokine into the extracellular space. The cells were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit in accordance with the manufacturer's protocol (eBiosciences, USA) and then stained directly with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC), and anti-mouse IL-10 (PerCP-Cyanine5.5) (eBiosciences, USA) for 30 min at 4°C. All these cell population were analyzed by Cytoflex S Flow Cytometer (Beckman Coulter, USA) and the data were analyzed by CytExpert software.
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