Tumor-bearing mouse lungs were collected from KP mice after which tumor nodules were excised and cut into about 1 mm pieces before placement under Hank’s Balanced Salt Solution (HBSS) containing 100 U/mL Collagenase D from Clostridium histolyticum (Sigma Aldrich) and 50 μg/mL DNase I grade II from bovine pancreas (Sigma Aldrich) for 40 minutes at 37°C. After digestion, cells were passed through a 70 µm strainer to remove clumps, and treated with ACK Lysing Buffer (Life Technologies). Cells were resuspended in FACS buffer (PBS + 2% fetal bovine serum) for flow cytometry. For multicolor flow cytometry, cells were first stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation (Life Technologies) for 30 minutes at 4 °C and washed twice with FACS buffer. Cells were treated with purified anti-mouse CD16/32 (BioLegend) for 15 minutes, and then antibody mixture was added. Thirty minutes later, the cells were washed twice with FACS buffer and fixed in 1% formalin or further processed for intracellular staining. For intracellular staining, cells were fixed/permeabilized with Foxp3/Transcription Factor Staining Buffer Set Kit (eBioscience) before antibodies were added. After two washes, samples were resuspended in FACS buffer before acquisition using BD LSR Fortessa or BD FACS Canto (BD Biosciences)].
Foxp3 transcription factor staining buffer set kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Foxp3/Transcription Factor Staining Buffer Set Kit is a laboratory tool designed for the detection and analysis of transcription factors, specifically Foxp3, within cells. The kit provides the necessary buffers and reagents to enable the staining and intracellular detection of these regulatory proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (3 mentions)
Facscanto, by BD (2 mentions)
Facsaria 3, by BD (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Collagenase d from clostridium histolyticum, by Merck Group (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd16 32, by BioLegend (1 mentions)
Intracellular fixation permeabilization buffer set kit, by Thermo Fisher Scientific (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Ly6c apc, by BD (1 mentions)
Ly6g v450, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Phosflow lyse fix buffer, by BD (1 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Phosflow perm buffer 3, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Cd8 alexa fluor 700, by BioLegend (1 mentions)
Foxp3 apc, by Thermo Fisher Scientific (1 mentions)
15 protocols using foxp3 transcription factor staining buffer set kit
1
Isolation and Analysis of Tumor Cells
2
Quantification of Regulatory T Cells
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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).
3
Multicolor Flow Cytometry Immunophenotyping
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Splenocytes were incubated with fluorescently labelled antibodies (CD11b, APC-eFluor 780, BD Bioscience, Franklin Lakes, NJ, USA; Ly6G, V450, BD Bioscience, Franklin Lakes, NJ, USA; Ly6C, APC, BD Bioscience, Franklin Lakes, NJ, USA) at 4 °C for 30 min. Following antibody incubation, cells were washed twice with FACS buffer (PBS, 10% FCS, 0.1% NaN3) before being fixed using the Foxp3/Transcription Factor Staining Buffer Set Kit (eBioscience, Carlsbad, CA, USA). Afterwards, samples were transferred into FACS buffer and analyzed using a LSR II flow cytometer (BD Bioscience) and Flow Jo Version 10 (Tree Star, Ashland, OR, USA). A total number of 50,000 events was analyzed for each sample.
4
Single-Cell Isolation and Staining
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The spleen and LNs were collected and single-cell suspensions of lymphocytes were prepared by mechanical disruption in 1640 medium supplemented with 2% FBS. Peripheral blood mononuclear cells (PBMC) were freshly isolated from healthy donors. Written informed consent was obtained for each participant according to institutional guidelines. All experiments were approved by the Fifth Medical Center of PLA General Hospital (permit number KY-2020-11-5-1). Single-cell suspensions were stained with the appropriate monoclonal antibody in phosphate-buffered saline containing 5% serum. To detect phosphorylated signaling proteins, NK cells were fixed with Phosflow Lyse/Fix buffer, followed by permeabilization with Phosflow Perm buffer III (BD) and staining with antibodies. All other intracellular proteins were stained according to the manufacturer’s instructions using Foxp3/Transcription Factor Staining Buffer Set Kit (eBioscience). Fortessa and FACSAria III (BD Biosciences, San Diego, USA) were used for analysis and cell sorting, with dead cells excluded by the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Carlsbad, USA). Primary antibodies used in flow cytometry were listed in Supplementary Table 4 .
5
Characterization of Pluripotent Stem Cells
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Single-cell suspensions of ESCs were obtained by dissociating with cell-dissociation buffer (Invitrogen) at 37°C for 20 min. Cells were washed twice with PBS and incubated with conjugated primary antibodies for 30 min on ice in the dark. Cells were washed once with PBS and resuspended for FACS analysis in PBS + 5% FBS. Flow cytometry was performed at the KU Leuven Flow Cytometry Facility using an FACS AriaIII (Becton Dickinson) or an FACS Canto (Becton Dickinson) for analysis. Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer Set kit (00-5523-00; Ebioscience), following the manufacturer's protocol. Antibodies are listed in the Supplemental Information .
6
Comprehensive Multicolor Flow Cytometry Analysis
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For the analysis of CTLs, splenocytes and MLNs cells were stained with fluorescently labelled antibodies (online supplementary table 4 ) including Fixable Viability Dye eFlour 780, PerCP-eFluor 710 anti-mouse CD3, FITC rat anti-mouse CD8a, PE rat anti-mouse TNF, BD Horizon BV421 hamster anti-Mouse CD279. To remove non-specific binding of fluorescently labelled antibodies to Fc receptors, the Fc receptors were blocked with purified anti-mouse CD16/32. For intracellular staining of TNF-α, the cells were treated with the Leucocyte Activation Cocktail for 12 hours. The cells were then fixed, permeabilised and washed using the Foxp3/Transcription Factor Staining Buffer Set Kit (#85-00-5523-00, eBioscience).
For the analysis of the macrophages, splenocytes and MLNs cells were stained with fluorescently labelled antibodies, including FITC rat anti-mouse CD11b, PE rat anti-mouse F4/80, PerCP-Cy5.5 anti-mouse CD16/32, anti-mouse CD279 (PD-1) and FcR block. Gates and quadrants were based on fluorescence minus one (FMO) control. The samples were detected with BD FACSVerse (BD Biosciences). The data were analysed by FlowJo_V10.
For the analysis of the macrophages, splenocytes and MLNs cells were stained with fluorescently labelled antibodies, including FITC rat anti-mouse CD11b, PE rat anti-mouse F4/80, PerCP-Cy5.5 anti-mouse CD16/32, anti-mouse CD279 (PD-1) and FcR block. Gates and quadrants were based on fluorescence minus one (FMO) control. The samples were detected with BD FACSVerse (BD Biosciences). The data were analysed by FlowJo_V10.
7
Flow Cytometry Analysis of Immune Cells
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All cells were incubated with Live/Dead Fixable Blue stain (#L23105, Thermo Fisher Scientific) for 15 minutes at 4 °C. Surface antibody staining (anti-human CD4, CD8, CD25) was performed for 30 minutes at 4 °C, followed by fixation/permeabilization for 30 minutes at 4 °C (FOXP3/Transcription Factor Staining Buffer Set Kit, #00-5523-00, eBioscience). Cells were then stained for intracellular factor FOXP3 for 30 minutes at room temperature. The following antibodies were used at a concentration of 5 µl in 100 µl staining volume: CD8-Alexa Fluor 700 (#344724, BioLegend), CD4-APC/Fire 750 (#300560, BioLegendCD25-BV510 (#302639, BioLegend), and FOXP3-APC (#17-4777-42, eBioscience). All samples were acquired with an LSRII flow cytometer and analyzed with FlowJo software (version 10.4.0) and a multistep gating strategy to identify immune cells (Supplementary Fig. 14 and Supplementary Fig. 15 ).
8
B Cell Activation and Phosphorylation
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The B cells (1 × 105 cells/mL per condition) were resuspended in a complete medium without serum and incubated for 10 min at 37 °C. Then, the B cells were stimulated with 10 µg/mL polyclonal anti-human IgM F(ab)2 fraction for 45 s in the presence or absence of each type of m/lEVs pool (B cell: m/lEVs ratio of 1:3). The cells were placed on an ice bath for 15 min and labeled with anti-human CD19 and CD38 monoclonal antibodies (Table S1 ). These cells were then fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set Kit (eBiosciences), as previously reported for phosphoproteins [31 (link)]. The B cells were labeled with anti-phosphotyrosine antibody (Table S1 ) in permeabilization buffer at 4 °C and incubated in the dark for 30 min. Pervanadate was used as the positive control of phosphorylation [32 (link)]. Fluorescence minus one was used as the staining control. The results obtained were presented as MFI.
9
Characterizing T-cell Subsets by Flow Cytometry
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The SPLCs were collected into FACs tubes and washed in PBS. Before staining‐stimulated SPLCs for analysis, restimulated lymphocytes were treated with 5 μg/mL brefeldin A (Biovision, C) for 6 h. Treg cells were stained with AlexaFluor 488‐anti‐CD4, PE‐cyanine7‐anti‐CD25, and stained further with PE‐anti‐Foxp3. Th17 cells were stained with AlexaFluor 488‐anti‐CD4 and AlexaFluor 647‐ anti‐IL‐17A (eBioscience). Acquisitions were performed using the Navios flow cytometer (Beckman Coulter). Foxp3 Transcription Factor Staining Buffer Set Kit (eBioscience) was used in fix and permeabilize cells. FlowJo10 software was used to measure the percentage of CD25+ /FOXP3+ Tregs and IL‐17+ Th17 cells in the CD4+ gate.
10
Multiparameter flow cytometry for immune cell analysis
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Isolated cells were resuspended in FACS buffer and incubated with Fc-block (Purified Rat Anti-Mouse CD16/CD32: isotype Rat IgG2b, clone: 2.4G2, BD Biosciences) for 20 min on ice to block non-specific antibody staining. Monoclonal antibodies for surface staining were subsequently added and incubated on ice for 30–40 min in concert with Live/Dead Aqua Stain (Thermo Fisher Scientific) to elucidate live cell populations. Intracellular staining for CXCR3 expression was performed using the Foxp3 Transcription Factor Staining Buffer Set Kit (eBioscience) as per the manufacturers’ instructions. Immediately before FACS acquisition, Flow-CountTM Fluorospheres (Beckman Coulter, Miami, FL, USA) were added to allow assessment of total cell counts. The antibodies used for flow cytometry are listed in Supplementary Table S2 . Flow cytometric analysis were performed using LSR Fortessa (BD Biosciences) flow cytometers with FACSDiva software (Becton Dickinson, Sparks, MD, USA). Data were exported and analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA).
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