Flow cytometry analysis was done on cells isolated from the spleen and small intestines. Splenocytes were separated through a 70 µm cell strainer. After washing and lysis of erythrocytes (10× Lysis Buffer: 1,5M NH4Cl, 100 mM KHCO3, 0,5M EDTA), 1×106 cells were preincubated with 1 µL FcR Blocking Reagent (130-092-575, MiltenyiBiotec) and stained for extracellular surface marker (CD4, CD8α and CD25; CD11b, Ly6g and Ly6c or CD4, CD8α, TCRβ and TCRγδ) at 4°C for 30 min. The Transcription Factor Staining Buffer Set (130-122-981, MiltenyiBiotec) was used for fixation and permeabilisation. Afterwards, the cells were again treated with FcR Blocking Reagent and stained for transcription factors (RORγt, GATA3, TBET and FOXP3) at 4°C for 30 min. Data evaluation was done by flow cytometry (BD, LSRII) and FlowJo (BD).
Transcription factor staining buffer set
Manufactured by Miltenyi Biotec
Sourced in Germany
The Transcription Factor Staining Buffer Set is a laboratory equipment designed for the detection and analysis of transcription factors in cells. It provides the necessary buffers and reagents to facilitate the staining and intracellular localization of transcription factors.
Automatically generated - may contain errors
Lab products found in correlation
Brilliant stain buffer, by BD (2 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Sp8 laser scanning microscope, by Leica (1 mentions)
Reafinity, by Miltenyi Biotec (1 mentions)
Zombie uv fixable viability kit, by BioLegend (1 mentions)
Aurora full spectrum flow cytometer, by Cytek (1 mentions)
Anti foxp3 pe, by Miltenyi Biotec (1 mentions)
Cell id intercalator ir, by Standard BioTools (1 mentions)
Cell id cisplatin, by Standard BioTools (1 mentions)
Helios system, by Standard BioTools (1 mentions)
Alexa fluor 488 conjugated antibody, by BioLegend (1 mentions)
Anti human cd4 bv510, by BD (1 mentions)
Anti human cd45ra apc, by BD (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
8 protocols using transcription factor staining buffer set
1
Multiparametric Flow Cytometry of Immune Cells
2
Imaging Stress-Induced β-Catenin Localization
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Cells (1 × 104 cells/plate) were seeded in a 35 mm glass bottom cell culture dish in 500 µL complete medium and incubated at 37 °C. Three days later, cells were induced for stress with IL-1β for HTB161 and with IL-1β+TNFα for HTB75. Treatments were given after 48 h of induction, for 16 h at sub-lethal concentrations. For fixation and permeabilization of cells, Transcription Factor Staining Buffer Set (130-122-981; Miltenyi Biotech, Bergisch Gladbach, Germany) was used, and cells were incubated for 30 min at 4–8 °C. For staining, β-Catenin monoclonal antibody (130-121-990-PE; REAfinity™ Miltenyi Biotech, Germany) was used, and cells were incubated for 40 min at 4–8 °C. Hoechst (EasyProbes™ 33342; ABP Bioscience, Beltsville, MD, USA) was used for nuclear staining. Cell microscopy and image acquisition was carried out using a Leica SP8 laser scanning microscope (Leica, Wetzlar, Germany), equipped with 405 and 552 nm solid state lasers, HC PL APO CS 63×/1.2 water immersion objectives (Leica, Wetzlar, Germany) and Leica Application Suite X software (LASX, Leica, Wetzlar, Germany). Hoechst, 5(6)-Carboxyfluorescein and PE (red)emission signals were detected with PMT and HyD (hybrid) detectors in ranges of 415–490 nm, and 565–660 nm, respectively.
3
Characterizing Regulatory T-cell Subsets
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Flow cytometry analysis was performed using a Cytek® Aurora full spectrum flow cytometer (Cytek Biosciences, California, USA). Cryopreserved PBMCs were gently thawed with a mean recovery of 87.50% viable cells, assessed by the Zombie UV™ Fixable Viability Kit (BioLegend, BioLegend, San Diego, CA, USA). 0.5 × 106 PBMCs were stained in duplicate by using anti-CD3 BV570 (UCHT1), anti-CD4 VioBright R720 (REA623), anti-CD25 PE-Vio615 (REA570), anti-CD45RA VioGreen (REA1047), anti-CD197 (CCR7) PE-Vio770 (REA108), anti-CD279 (PD-1) VioBright 515 (REA1165), and anti-FoxP3 PE (REA1253) (Miltenyi Biotec, Bergisch Gladbach, Germany). The Transcription Factor Staining Buffer Set (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for nuclear staining. Isotype controls were used to rule out unspecific binding of antibodies. Gating was performed using FlowJo™ 10.8.1 (see Fig. 2 for gating strategy). Tregs were gated as CD4+CD25highFoxP3+, PD-1+ effector Tregs (eTregs) as CD4+CD25highFoxP3+CD45RA−PD-1+, and naïve Tregs (nTregs) as CD4+CD25highFoxP3+CD45RA+CCR7+. Median fluorescence intensity (MFI) was calculated in FlowJo™ and used as a relative surrogate marker of protein level.![]()
Gating strategy
4
Comprehensive PBMC Immunophenotyping Protocol
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PBMC from patients were thawed. Briefly, cells were stained 5 minutes in RPMI supplemented with 0.5 μM Cisplatin Cell-ID (Fluidigm, San Francisco, CA) in RPMI 1640 before washing with 10% FCS in RPMI 1640. Cell pellets were resuspended in 80μl of 0.5% BSA in PBS. Then 60 μl of each surface staining cocktail, lymphoid or myeloid, were added to 40μl of resuspended cells. After staining, cells were washed in 0.5% BSA in PBS before fixation/permeabilization with the transcription factor staining buffer set (Miltenyi, Bergisch-Gladbach, Germany). Then 60μl of each surface staining cocktail, lymphoid or myeloid, were added to 40μl of resuspended cells in Perm Buffer. The panel of antibodies is listed in Table S3 and in Key resources table . After intracellular staining, cells were washed twice before staining in DNA intercalator solution (2.5% Paraformaldehyde, 1:3200 Cell-ID Intercalator-Ir (Fluidigm, San Francisco, CA) in PBS). Samples were cryopreserved at −80°C until acquisition on Helios System (Fluidigm, San Francisco, CA).
5
Phenotypic Characterization of Regulatory T Cells
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PBMCs isolated by Ficoll-Paque gradient centrifugation from mutation carrier and sex-matched controls were thawed and plated 3 x 106/ml in RPMI 1640 medium. The cells were allowed to rest for 2 hours and thereafter live and dead cells were stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen; L10119). The cells were washed twice in PBS (Sigma Aldrich; D1408) with 2% FBS and centrifuged at 550 x g for 2 minutes. The cells were stained for surface markers in Brilliant Stain Buffer (BD Biosciences; 563794) for 30 minutes at 4°C, washed, fixed and permeabilized with Transcription Factor Staining Buffer Set (Miltenyi Biotec, 130-122-981) for 30 minutes at 4°C. After permeabilization the cells were washed and stained for FOXP3 for 35 minutes at 4°C. The antibodies against anti-human CD4 BV510 (562970), anti-human CD127 PE Cy7 (560822), anti-human CD45RA APC (561210), anti-human CD25 BV421 (BD Biosciences; 564033), anti-human CCR7 PE (566741), anti-human CCR6 PE (551773) and anti-human CXCR3 PE (550633) were purchased from BD Biosciences. The Alexa Fluor 488 conjugated antibody against human FOXP3 was purchased from Biolegend (Biolegend; 320112) The data was collected with BD LSRFortessa™ using BD FACSDiva software and analyzed with FlowJo™ 10 (BD Biosciences).
6
Quantification of FOXP3+ Treg cells
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PBMCs from patient and healthy controls were plated at 3 × 106/ml in supplemented RPMI 1640 medium (2.6). The cells were allowed to rest overnight and stained for live and dead cells. The cells were stained for surface markers in Brilliant Stain Buffer (BD Biosciences; 563,794) for 30 min at 4 °C, washed, fixed and permeabilized with Transcription Factor Staining Buffer Set (Miltenyi Biotec; 130-122-981) for 30 min at 4 °C. After permeabilization the cells were washed and stained for FOXP3 for 35 min at 4 °C. The experiment was performed twice with two and four healthy controls, all stained samples were done in duplicates.
7
Quantifying Transcription Factor KLF2 in PBMCs
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CPT-isolated PBMCs from mutation carriers and sex-matched controls were thawed and plated 3 x 106/ml in RPMI 1640 medium. The cells were allowed to rest overnight and thereafter live and dead cells were stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen; L10119). The cells were washed twice in PBS (Sigma Aldrich; D1408) with 2% FBS and centrifuged at 550 x g for 2 minutes. The cells were stained for surface markers for 30 minutes at 4°C, washed, fixed and permeabilized with Transcription Factor Staining Buffer Set (Miltenyi Biotec, 130-122-981) for 30 minutes at 4C°. After permeabilization the cells were washed and stained for KLF2 for 35 minutes at 4C°. The antibodies against anti-human CD3 Alexa Fluor 488 (557694), anti-human CD4 PE Cy7 (557852) were purchased from BD Biosciences. The PE conjugated antibody against human KLF2 was purchased from Miltenyi Biotec (Miltenyi Biotec; 130-111-039). The data was collected with BD LSRFortessa™ using BD FACSDiva software and analyzed with FlowJo™ 10 (BD Biosciences).
8
Multiparametric Analysis of Splenocyte Phenotypes
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One million splenocytes were labeled for 30 minutes at 4°C with antibodies listed in Table S1. Before analysis, DAPI (Sigma-Aldrich) was added to exclude dead cells. For intracellular staining, cells were first labelled with LIVE/DEAD TM Fixable Violet Dead Cell Stain Kit (ThermoFisher Scientific, L34955) according to the manufacturer's recommendations. Then, extracellular staining was performed with the required antibodies. Finally, a transcription factor staining buffer set (Miltenyi, 130-122-981) was used to fix and permeabilize cells before intracellular staining with anti-Ki-67 antibody. For apoptosis analysis, the cells were stained with FITC-AnnexinV (Tau Technologies, A700) and DAPI (Sigma Aldrich). AnnexinV + DAPI -cells were considered apoptotic and AnnexinV + DAPI + cells were considered dead. All tubes were analyzed on the CytoFlex (Beckman Coulter). Gating strategies are displayed in Fig. S1. Flow cytometry files were analyzed using FlowJo software v10.6.2.
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