Used fluorochrome-conjugate monoclonal antibodies included: CD301 (Milteny Biotech, clone: REA687), CD11b (Biolegend, clone: M1/70), F4/80 (Biozol, clone: BM8), TIM4 (BioLegend, clone: F31–5G3), CD45.1 (BioLegend, clone: A20). Staining was performed in 1% fetal bovine serum in PBS for 30 min on ice, including anti-CD16/CD32 (Biozol); dead cells were excluded with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo scientific). For intracellular RELMα staining, cells were processed with the Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) following the manufacturer protocol. RELMα primary antibody staining (Peprotech Cat# 500-P214) was detected using Alexa Fluor-421 anti-rabbit secondary antibody (Life technologies). Tagged ETV1 were labeled using a monoclonal anti-V5 antibody (Life Technologies, RRID: AB_2556564) and detected with an anti-Mouse IgG2a PE secondary (eBioscience). EdU staining was performed using the Click-iT® EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Dead and Apoptotic cells were detected using Annexin V, Alexa Fluor 550 conjugate (Thermo Fisher Scientific) following the manufacturer’s protocol. Cells were analyzed using LSR Fortessa flow cytometers (BDBiosciences) and data were processed using FlowJo software (FlowJo).
Transcription factor staining buffer set
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Transcription Factor Staining Buffer Set is a product designed for the detection and analysis of transcription factors in cell samples. It provides the necessary reagents for the permeabilization, fixation, and staining of cells to allow for the intracellular detection of transcription factors by flow cytometry or other analysis methods.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (9 mentions)
Ionomycin, by Merck Group (7 mentions)
Golgistop, by BD (7 mentions)
Phorbol myristate acetate (pma), by Merck Group (6 mentions)
Brefeldin a, by Merck Group (5 mentions)
Dnase 1, by Merck Group (4 mentions)
Facscanto, by BD (4 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Pe cy7, by BD (3 mentions)
Fcr blocking reagent, by Miltenyi Biotec (3 mentions)
Facscalibur, by BD (3 mentions)
Lsrii instrument, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Fc block, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Facsaria 2, by BD (3 mentions)
Ic fixation buffer and permeabilization buffer, by Thermo Fisher Scientific (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
101 protocols using transcription factor staining buffer set
1
Multiparametric Flow Cytometry of Immune Cell Subsets
2
Characterization of RBD-Specific B Cells
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Biotinylated RBD protein (Acro Biosystems, cat# SPD-C82E9) was complexed with fluorochrome-conjugated streptavidin APC (BioLegend, cat# 405207) to yield RBD-Tetramers. Lymph node cells from FNA were incubated with the RBD-fluorochrome complex and subsequently stained with aqua live/dead dye (Invitrogen, L34957), anti-human IgM (FITC, clone: G20–127, BD cat# 555782, 1:5), anti-human IgG (PE-Cy7, clone: G18–145, BD cat# 561298, 1:20), anti-human CD3 (AF700, clone: SP34-2, BD cat# 557917, 1:20), anti-human PD-1 (BV650, clone: EH12.1, BD cat# 564104, 1:20), anti-human CD20 (PE/Dazzle 594, clone: 2H7, BioLegend cat# 302348, 1:20), anti-human CD4 (APC-Cy7, clone: OKT4, BioLegend cat# 317418, 1:20) and anti-human CXCR5 (PerCP-eF710, clone: MU5UBEE, ThermoFisher cat# 46-9185-42, 1:20). Cells were fixed/permeabilized using Transcription Factor Staining Buffer Set (ThermoFisher, cat# 00-5521-00) and further stained with anti-human Bcl-6 (PE, clone: 7D1, BioLegend cat# 358504, 1:20) and anti-human Ki-67 (BV421, clone: 11F6, BioLegend cat# 151208, 1:20). Sample acquisition was performed on a BD FACS Symphony and data were analyzed with BD FlowJo V10 software.
3
Immune Cell Phenotyping by Flow Cytometry
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Single cell suspensions were prepared from the spleen, lymph nodes and mesenteric lymph nodes by tissue digestion in the presence of collagenase D (1 mg/ml, Sigma Aldrich) and DNase1 (20 μg/ml, Sigma Aldrich) at 37°C for 30 min and filtration through a cell strainer 70 μm (Thermo Fisher). Cell suspensions were subjected to red blood cell lysis, washed, and stained with unconjugated and conjugated fluorescent antibodies listed in Table S1 . Intracellular staining for Foxp3 and BCL6 were performed using the transcription factor staining buffer set (Thermo Fisher) according to the manufacturer's instructions. For optimal resolution of CXCR5 across CD4+ T cell subsets, staining with CXCR5 biotinylated antibody to identify CD4+ T follicular helper (Tfh) cells was performed for 1 h at room temperature, prior to conventional staining with other relevant markers. Samples were acquired on an Attune flow cytometer (Thermo Fisher) and analyzed using FlowJo software (Tree Star).
4
Flow Cytometric Analysis of Cryopreserved and Freshly Isolated PBMCs
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Cryopreserved PBMC samples of patients and controls were thawed, processed, stained, and analyzed by flow cytometry together. Freshly isolated PBMC from COVID patients and COVID-negative controls were stained and analyzed together on the day of sampling. PBMC were stained in 50μl of PBS, 2mM EDTA, 10% FCS (v/v) containing FcR blocking reagent (Miltenyi Biotec) with fluorescently labeled antibodies as indicated in S2 Table and incubated for 30 min at 4°C. Fixable viability dyes were used according to the manufacturers’ protocol. Cells were fixed with BD Cytofix (Cat. # 554655), washed and resuspended in PBS. Intracellular staining for Ki67 was performed using the Transcription Factor Staining Buffer Set (ThermoFisher, cat. # 00-5523-00) following the manufacturers instructions. Samples were measured using the Cytek Aurora (Cytec Biosciences) with the recommended Cytek assay settings, where gains are automatically adjusted after each daily QC based on laser and detector performance to an optimal value, ensuring comparability between measurements. Cells from coculture experiments were measured using the CytoFLEX S flow cytometer (Beckman Coulter). FCS files were exported and analyzed with FlowJo software v10.7.1.
5
Quantifying Immune Cell Surface Markers
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Cell surface staining of blood mononuclear cells and isolated monocytes was carried out by incubation with FITC antihuman CD14 and APC anti human CD14 (BioLegend, San Diego, CA, US). Expression of protein levels of IRAK-M was determined by flow cytometry intracellular staining. THP-1 cells and PBMCs were fixed and permeabilized (Transcription Factor Staining Buffer Set, Thermo Fisher Scientific, Waltham, MA, US). Then, staining was carried out with anti-human IRAK-M rabbit polyclonal antibody (ab-8116, Abcam) and anti-rabbit APC conjugated secondary antibody (BioLegend, San Diego, CA, US). The proper isotype control was used as a control. The flow cytometry events were acquired in a FACS Calibur (BD Biosciences, San Jose, CA) and analyzed with the use of Summit v4.3 Software.
6
Multi-parameter Flow Cytometry Analysis of Immune Cells
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The spleen was dissociated into single cells by mashing the tissue through a
70-µm cell strainer. Red blood cells from spleens were lysed using 0.83% (w/v)
of ammonium chloride. For the detection of immune cell subtypes, the following
antibodies were used: Alexa Fluor 647 anti-mouse FOXP3 (Biolegend, 126407),
Alexa Fluor 700 anti-mouse CD45 (Biolegend, 103127), Brilliant Violet 510
anti-mouse CD8a (Biolegend, 100751), Brilliant Violet 650 anti-mouse CD19
(Biolegend, 115541), Brilliant Violet 785 anti-mouse Ly-6C (Biolegend, 128041),
FITC anti-mouse CD3 (Biolegend, 100203), Pacific Blue anti-mouse CD4 (Biolegend,
100427), PE anti-mouse IL-4 (Biolegend, 504103), PC/Cy7 anti-mouse IFNgamma
(Biolegend, 505825), PE/Dazzle 594 anti-mouse IL-17A (Biolegend, 506937), and
PerCP/Cy5.5 anti-mouse CD11b (Biolegend, 101227). Viable cells were gated using
the Zombie NIR Fixable Viability Kit (Biolegend, 423105). For cell surface
staining, cells were incubated with 10% rat serum for 15 min prior to staining
with fluorescently labeled antibodies for 15 min on ice. For intracellular
staining, cells were fixed, permeabilized, and stained using the Transcription
Factor Staining Buffer Set (Thermo Fisher Scientific) according to the
manufacturer’s protocol. Flow cytometry was carried out and analyzed on a BD
LSRFortessa (BD Biosciences).
70-µm cell strainer. Red blood cells from spleens were lysed using 0.83% (w/v)
of ammonium chloride. For the detection of immune cell subtypes, the following
antibodies were used: Alexa Fluor 647 anti-mouse FOXP3 (Biolegend, 126407),
Alexa Fluor 700 anti-mouse CD45 (Biolegend, 103127), Brilliant Violet 510
anti-mouse CD8a (Biolegend, 100751), Brilliant Violet 650 anti-mouse CD19
(Biolegend, 115541), Brilliant Violet 785 anti-mouse Ly-6C (Biolegend, 128041),
FITC anti-mouse CD3 (Biolegend, 100203), Pacific Blue anti-mouse CD4 (Biolegend,
100427), PE anti-mouse IL-4 (Biolegend, 504103), PC/Cy7 anti-mouse IFNgamma
(Biolegend, 505825), PE/Dazzle 594 anti-mouse IL-17A (Biolegend, 506937), and
PerCP/Cy5.5 anti-mouse CD11b (Biolegend, 101227). Viable cells were gated using
the Zombie NIR Fixable Viability Kit (Biolegend, 423105). For cell surface
staining, cells were incubated with 10% rat serum for 15 min prior to staining
with fluorescently labeled antibodies for 15 min on ice. For intracellular
staining, cells were fixed, permeabilized, and stained using the Transcription
Factor Staining Buffer Set (Thermo Fisher Scientific) according to the
manufacturer’s protocol. Flow cytometry was carried out and analyzed on a BD
LSRFortessa (BD Biosciences).
7
Modulation of CD4+ T Cell Responses by Dendritic Cells
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CD4+ T cells from day 0 (unstimulated) and day 14 (PHA stimulated) were labelled with e450 proliferation dye (10 µM; ThermoFisher) and cultured with either: iDCs or mDCs at a ratio of 100,000 CD4+ T cells: 10,000 DCs in the absence (complete media) or presence of α-PD-1 (pembrolizumab, 1.0 μg/ml, UHB Pharmacy), IgG4 isotype control (1.0 μg/ml, Biolegend, 403702), α-CTLA-4 (ipilimumab, 1.0 μg/ml, UHB Pharmacy), or IgG1 isotype control (1.0 μg/ml, Biolegend, 403502) for 6 days. After 6 days culture, supernatants were analysed for IFNγ and IL-10 levels by ELISA (R&D Systems, DY285B, DY217B respectively). Cells were stimulated for 4 h with either brefeldin alone (10 µg/ml; Sigma-Aldrich), brefeldin + phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich) or brefeldin + PMA + ionomycin (750 ng/ml; Sigma-Aldrich). Cells were then washed, surface labelled with anti-human CD4 BV510 (Biolegend; 317444), fixed and permeabilised using transcription factor staining buffer set (ThermoFisher, 00-5523-00) and labelled with anti-human IFNγ PE and IL-10 APC (Biolegend; 506507; 506807 respectively). Cells were then analysed using a Cytek flow cytometer.
8
Multiparametric flow cytometry analysis of immune cells
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For analysis, single-cell suspensions were prepared by mechanical disruption of spleens unless specified otherwise. Viability was assessed by staining 1 × 108 cells/ml with fixable viability dye eFluor 780 (Thermo Fisher Scientific) for 20 min at 4°C. Fc receptors were blocked using anti-CD16/CD32 (BioXCell). For intracellular staining, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Surface or intracellular staining was performed at 1 × 108 cells/ml for 20 min at 4°C. The following antibodies were used: anti-CD4 BUV395 (GK1.5), anti-PD-1 BV421 (RMP1-30), anti-CD95 PE-Cy7 (Jo2), and anti-Bcl6 PE (K112-91) (BD Biosciences); anti-B220 PerCP-eFluor 710 (RA3-6B2), anti-CXCR5 biotin (SPRCL5), Streptavidin PE-Cy7, anti-ICOS FITC (7E.17G9), anti-Foxp3 APC (FJK-16s), anti-B220 eFluor 450 (RA3-6B2), and anti-CD25 PE (PC61.5) (Thermo Fisher Scientific); and anti-NFATc1 PE (7A6) and anti-GL7 FITC (GL7) (BioLegend). To identify influenza-specific B cells, we used tetramerized recombinant nucleoproteins conjugated with APC or PE (Flu tetramer) provided by Dr. Troy Randall (Allie et al, 2019 (link)). Data were acquired using a BD LSRFortessa and analyzed using FlowJo v10 (BD Biosciences).
9
Multiparametric Flow Cytometry Analysis
10
Flow Cytometry Analysis of Phosphatidylserine Exposure
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Antibodies and staining reagents are listed in Table S2 . As PS‐staining reagents we used the previously published recombinant MFG‐E8‐eGFP (Kranich et al., 2020 (link); Trautz et al., 2017 (link)) and mC1‐biotin multimerised with streptavidin‐AF647 for flow cytometry or streptavidin‐gold for TEM. mC1‐multimers are commercially available through BioLegend as ApotrackerTM Tetra reagents. In flow cytometry. All PS‐staining reagents are Ca2+ independent, highly stable and PS‐specific. Freshly isolated PBMCs were incubated with Fc block before live/dead and cell surface antibody staining. PBMCs surface staining was performed in staining buffer (PBS containing 2% of fetal calf serum) for 25 min on ice. Then cells were washed and fixed with 4% PFA for 30 min at room temperature (RT), washed again, filtered and analysed on an ImageStreamx MKII imaging flow cytometer (Luminex). Samples were acquired with low flow rate/high sensitivity and 60× magnification. Images were acquired using bright field illumination and the excitation lasers 405, 488, 561 and 642 nm.
For intranuclear Ki‐67 staining, cells were fixed and permeabilised using the Transcription Factor Staining Buffer Set (ThermoFisher, cat. #00‐5523‐00) according to the manufacturers' instructions. Intranuclear staining was performed in permeabilisation buffer for 30 min at RT.
For intranuclear Ki‐67 staining, cells were fixed and permeabilised using the Transcription Factor Staining Buffer Set (ThermoFisher, cat. #00‐5523‐00) according to the manufacturers' instructions. Intranuclear staining was performed in permeabilisation buffer for 30 min at RT.
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