PBMC were plated and stimulated with CpG-ODN 2006 (Aurogene Srl) alone or in the presence of 100 μl of CM-hAMSC as described above. After 5 days of culture, cells were collected and protein expression of the transcription factors BCL6, PAX-5, IRF-4, and BLIMP-1 was analyzed by flow cytometry. After exclusion of dead cells by eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific) staining, cells were stained for 20 min at 4°C with CD19 BB700 (SJ25C1, 1:200), CD3 BV510 (UCHT1, 1:100), CD14 BV510 (MΦP9, 1:200), CD27 PE-Cy7 (M-T271, 1:100), all from BD Biosciences. After washing in stain buffer, the cells were fixed and permeabilized with Transcription Factor Buffer Set (BD Biosciences) for 40 min at 4°C, according to the manufacturer's instructions. Then, cells were stained for 40 min at 4°C with specific antibody against BCL6 BV421 (K112-91, 1:30), PAX-5 PE (1H9, 1:100), IRF-4 PE (Q9–343, 1:100), BLIMP-1 Alexa Fluor® 647 (6D3, 1:100). Finally, the cells were washed with Transcription Factor Buffer Set (BD Biosciences), acquired on a FACSAria III (BD Biosciences), and analyzed using FCS express v5.0 (DeNovo Software, Los Angeles, CA, USA).
Transcription factor buffer set
Manufactured by BD
Sourced in United States, Canada
The Transcription Factor Buffer Set is a collection of buffers designed for the study of transcription factors. The set includes buffers formulated to maintain the stability and activity of transcription factors during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (11 mentions)
Lsrfortessa, by BD (11 mentions)
Ionomycin, by Merck Group (9 mentions)
Facscalibur, by BD (8 mentions)
Facscalibur flow cytometer, by BD (7 mentions)
Fixable viability stain 780, by BD (7 mentions)
Golgiplug, by BD (6 mentions)
Cytofix cytoperm kit, by BD (6 mentions)
Fixation permeabilization kit, by BD (5 mentions)
Facscanto 2 flow cytometer, by BD (5 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (5 mentions)
Fc block, by BD (5 mentions)
Facsdiva software, by BD (5 mentions)
Golgistop, by BD (5 mentions)
Brilliant stain buffer, by BD (5 mentions)
Fixation permeabilization solution kit, by BD (4 mentions)
Facsaria 2, by BD (4 mentions)
Celltrace violet, by Thermo Fisher Scientific (4 mentions)
Brefeldin a, by Merck Group (4 mentions)
Near ir dead cell stain kit, by Thermo Fisher Scientific (4 mentions)
206 protocols using transcription factor buffer set
1
Flow Cytometry Analysis of B Cell Transcription Factors
2
Multicolor Flow Cytometry Staining
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Prior to antibody staining, dead cells were labelled with Live/Dead Fixable Near‐IR Dead Cell Stain Kit (Excitation 750, Emission 775; ThermoFisher Scientific) for 15 min at room temperature and excess dye washed away. Following the wash, antibodies for staining surface targets were diluted in fluorescence‐activated cell sorting (FACS) buffer and applied to cells for 30 min on ice. Unattached surface antibodies were removed with a wash. A Transcription Factor Buffer Set (BD Pharmigen) optimised for staining of intracellular targets for flow cytometric analysis was used to fix/permeabilise cells for 45 min at 4°C. Afterward, cells were washed twice in a perm/wash buffer from the same set. Antibodies for intracellular targets were diluted in perm/wash buffer, applied to cells for 45 min at 4°C, and washed twice prior to analysis on an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific). All analyses were done using FlowJo Cytometric Software (TreeStar, Inc.).
3
Flow Cytometry Analysis of Tonsillar Cells
4
Coexpression Analysis of NGN3 and CD133
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To determine coexpression of NGN3 and CD133, single cell suspensions of exocrine tissue were divided in two aliquots. For cytospin analysis, one aliquot of cells was immunomagnetically enriched for CD133 then stained with mouse anti-CD133-PE at 4°C for 10 min., fixed in 2% PFA for 10 min. at room temperature and spun onto a slide using a cytospin. Slides were washed in 50 mM glycine/PBS, blocked with 5% donkey serum 1% BSA 0.1% triton X100, then stained with rabbit anti-human NGN3 or rabbit Ig (isotype negative control) diluted in blocking buffer overnight at 4°C. Proteins were visualized with donkey anti-rabbit Alexa Fluor 647 secondary antibody (Life Technologies, Carlsbad, CA, USA) and PE. Nuclei were stained with Hoechst 33342. For FACS analysis, the second aliquot of cells was stained with mouse anti-CD133-PE or IgG2b isotype negative control at 4°C for 10 min, then fixed, blocked and permeabilized using Transcription Factor Buffer Set (BD Pharmigen, San Jose, CA, USA) reagents. Cells were then stained with rabbit anti-human NGN3 or rabbit Ig then stained with anti-rabbit Alexa Fluor 647. Cell population was gated on FL2/SSC to identify CD133+ then analyzed for NGN3 expression in FL4.
5
Multicolor Flow Cytometry Staining
6
Intracellular Profiling of Organoid Differentiation
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FACS intracellular staining: Organoids during the 2 nd ,5 th ,9 th and 14 th week of differentiation were dissociated into single cells by incubating them till dissociating at 37 o C within papain solution under shaking conditions. Then, the cells pelleted at 400xg for 5min and followed incubation with TryplE Select for 10min to ensure dissociation into singe cells. Further, the cells passed through 70μM
(2 nd week) -100μM (5 th , 9 th , 14t h week) cell strainer to avoid aggregates. For both digesting steps 10% FBS/DMEM-F12 as digesting deactivation solution applied to the cells. Then, for intracellular flow cytometric staining analysis the Transcription Factor Buffer set (BD Pharmigen) was applied and the cells were analyzed using flow cytometer (BD Biosciences FACS ARIAII).
Primary antibodies used in this study: anti-PAX7, anti-MYOD1, anti-Pax3 in total amount of 400μg per staining, while as secondaries the Rhodamine RedTM-X (RRX) AffiniPure Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific (Jackson Immunoresearch Laboratories), Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2a specific (Jackson Immunoresearch Laboratories) in 1:50 dilution. As isotype controls, Mouse IgG1 kappa Isotype Control (Invitrogen, clone P3.6.2.8.1), Mouse IgG2a kappa Isotype Control, (Invitrogen, clone eBM2a) were used at 400μg total amount per staining.
(2 nd week) -100μM (5 th , 9 th , 14t h week) cell strainer to avoid aggregates. For both digesting steps 10% FBS/DMEM-F12 as digesting deactivation solution applied to the cells. Then, for intracellular flow cytometric staining analysis the Transcription Factor Buffer set (BD Pharmigen) was applied and the cells were analyzed using flow cytometer (BD Biosciences FACS ARIAII).
Primary antibodies used in this study: anti-PAX7, anti-MYOD1, anti-Pax3 in total amount of 400μg per staining, while as secondaries the Rhodamine RedTM-X (RRX) AffiniPure Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific (Jackson Immunoresearch Laboratories), Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2a specific (Jackson Immunoresearch Laboratories) in 1:50 dilution. As isotype controls, Mouse IgG1 kappa Isotype Control (Invitrogen, clone P3.6.2.8.1), Mouse IgG2a kappa Isotype Control, (Invitrogen, clone eBM2a) were used at 400μg total amount per staining.
7
Monocyte NLRP3 Inhibition Assay
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Monocytes from randomly selected aSAH patients at day 1 (n = 5) and NC (n = 4) obtained as described above were used for NLRP3 inhibition assays. A total of 0.5 × 106 monocytes per well were cultured in M6 plates and treated or not with 5 μM MCC-950 (INH-MCC, Ibian Technologies S.L). Cells were incubated at 37 °C and 5% CO2 for 16 h and then both monocytes and supernatants were collected for further analysis. Monocytes were washed two times with PBS and then treated following a standard protocol using the Transcription Factor Buffer Set (Becton–Dickinson Biosciences). Cells were labeled (30 min, 4 °C) with the anti-CD14, anti-NLRP3, and anti-ASC antibodies detailed in Table e1 . Additionally, caspase-1 activity was detected using the FAM FLICA caspase-1 kit following the manufacturer’s protocol (Bio-Rad Laboratories, Inc.). Cells were acquired using BD FACS-Calibur flow cytometer (Becton–Dickinson Biosciences), and data were analyzed using FlowJo vX.0.7 software (FlowJo). Gating schemes are showed at Fig. e1 . Supernatants were stored at − 80 °C until analysis by ELISA (IL-18, GSDMD and TF) according to manufactures’ instructions as available in Table e2 or BD Human Inflammatory Cytokine CBA kit (IL-1β), as previously described.
8
Multiparametric Flow Cytometry Analysis of MAIT Cells
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For extracellular staining, cultured PBMCs were incubated with FITC-anti-CD3 antibody (Catalog #300440, BioLegend, Inc., San Diego, CA, USA), APC-anti-CD4 antibody (Catalog #300514, BioLegend), APC/Cyanine7-anti-CD8 antibody (Catalog #344714, BioLegend), PE-anti-CD161 antibody (Catalog #339904, BioLegend), and PE/Cyanine7-anti-TCR Vα7.2 antibody (Catalog #351712, BioLegend) for 15 minutes at 25°C. To detect intracellular cytokines, cells whose surfaces were stained by markers of MAIT cells were then fixed and permeabilized in Transcription Factor Buffer Set (Catalog #562574, Becton Dickinson and Co., Franklin Lakes, NJ, USA) at 4°C in accordance with the instructions of the manufacturer. Intracellular protein probing was performed by staining with BV421-anti-IL-17A antibody (Catalog #512322, Becton Dickinson and Co.) and BV650-anti-IFN-γ antibody (Catalog #506542, Becton Dickinson and Co.) for 30 minutes at 4°C. Cells were washed and resuspended in phosphate-buffered saline prior to flow cytometric analysis.
9
MDSC Phenotype and Function Analysis
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Surface expression of the PD-L1 molecule on MDSCs was assessed using PE-labeled anti-PD-L1 monoclonal antibodies (BD PharMingen, USA) in PMN-MDSCs (Lin–HLA-DR–CD33+CD66b+), E-MDSCs (Lin−HLA-DR–CD33+CD66b–) and M-MDSCs(14+HLA-DRlow/–) according to standard flow cytometric approaches for surface antigens.
To assess the intracellular expression of Arg-1 and IDO, PBMCs were incubated with fluorochrome-conjugated monoclonal antibodies to PMN-MDSCs, E-MDSCs, and M-MDSCs as described above. Next, the cells were permeabilized using Transcription Factor Buffer Set (Becton Dickinson, USA) and stained with PE-conjugated anti-Arginase-1 (BD PharMingen, USA) or PE-conjugated anti-IDO (BD PharMingen, USA) monoclonal antibodies. Relative counts of MDSCs expressing Arg-1, IDO, and PD-L1 were assessed among Lin–HLA-DR–CD33+CD66b+, Lin–HLA-DR–CD33+CD66b–, and CD14+HLA-DRlow/– cells. Isotype antibodies conjugated with the same fluorochromes were used as a negative control.
To assess the intracellular expression of Arg-1 and IDO, PBMCs were incubated with fluorochrome-conjugated monoclonal antibodies to PMN-MDSCs, E-MDSCs, and M-MDSCs as described above. Next, the cells were permeabilized using Transcription Factor Buffer Set (Becton Dickinson, USA) and stained with PE-conjugated anti-Arginase-1 (BD PharMingen, USA) or PE-conjugated anti-IDO (BD PharMingen, USA) monoclonal antibodies. Relative counts of MDSCs expressing Arg-1, IDO, and PD-L1 were assessed among Lin–HLA-DR–CD33+CD66b+, Lin–HLA-DR–CD33+CD66b–, and CD14+HLA-DRlow/– cells. Isotype antibodies conjugated with the same fluorochromes were used as a negative control.
10
Monocyte Characterization in aSAH Patients
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Monocytes from aSAH patients at day 1 (n = 28) (0–24 h), day 2–5 (n = 18) (48–120 h), and day 7–10 (168–240 h) (n = 18) after aSAH event and from NC (n = 14) were washed two times with PBS and then treated following a standard protocol using the Transcription Factor Buffer Set (Becton–Dickinson Biosciences). Cells were labeled (30 min, 4 °C) with the anti-CD14, anti-NLRP3, and anti-ASC antibodies detailed in Table e1 . Additionally, caspase-1 activity was detected using the FAM FLICA caspase-1 kit following the manufacturer’s protocol (Bio-Rad Laboratories, Inc.). Cells were acquired using BD FACS-Calibur flow cytometer (Becton–Dickinson Biosciences), and data were analyzed using FlowJo vX.0.7 software (FlowJo). Gating schemes are showed at Fig. e1 .
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