Cells deriving from digested muscles or cultured-myogenic cells were labeled with 1 mL of Fixable Viability Stain 780 (1:1,000, BD Biosciences) in HBSS for 10 min. Cells were washed with HBSS, and then fixed and permeabilized in 500 μL of fixation solution (Transcription Buffer Set, BD Pharmingen) for 45 min at +4°C, accordingly to manufacture instructions. Fixed cells were washed with the permeabilization/wash buffer (Transcription Buffer Set, BD Pharmingen) and proceeded for nuclear immunostaining. Cells were incubated with BSA 5% for 15 min and then with the mix of conjugated antibodies for 50 min at room temperature. The following mix of conjugated antibodies was used: mouse anti-PAX3/7-AF647 (B-5), mouse anti-MYOD-PE coupled using SiteClick PE Antibody Kit (no. S10467, Invitrogen), mouse anti-MYOG-AF488 (5FD), and mouse anti-KI67-bV421. A detailed list of the coupled antibodies and the working dilutions used is provided in Table S3 . Cells were washed with the permeabilization/wash solution, re-suspended in HBSS and analyzed using a BD FACSCanto Flow Cytometer (BD Biosciences). For in vivo experiments, acquisitions were performed on a mean of 35,000 living cells for postnatal muscles and of 5,000 living cells for adult muscles. For in vitro experiments acquisitions were performed on 5,000 to 10,000 living cells.
Transcription buffer set
Manufactured by BD
Sourced in United States
The Transcription Buffer Set is a collection of buffers designed for use in transcription-related laboratory procedures. The set includes specific buffers for optimal performance in transcription experiments, providing the necessary components to facilitate the transcription process. The set is intended to support researchers in their investigations involving gene expression and related molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Fixable viability stain 780, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Ctla4 apc, by BD (1 mentions)
Anti cd16 32, by BioLegend (1 mentions)
Horizon v450, by BD (1 mentions)
Horizon v500, by BD (1 mentions)
Lsrfortessa machine, by BD (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Kaluza flow cytometry analysis software version 2, by Beckman Coulter (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Yellow arc qdot585, by Thermo Fisher Scientific (1 mentions)
Cd16 32 fc block, by BD (1 mentions)
Facs aria fusion flow cytometers, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
8 protocols using transcription buffer set
1
Multiparametric Flow Cytometry of Myogenic Cells
2
Treg Subpopulations and CTLA-4 Expression
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Flow cytometry was used to determine the lymphocyte subtypes in the clBALF, hlBALF and PB. The numbers of Treg subpopulations and Tregs with the presence of CTLA-4 in samples were determined by a panel of monoclonal antibodies against: CD4 PE-Cy7, CD25 PE, CD127 BV421, Foxp3 Alexa Fluor 488, CTLA-4 APC (BD, USA); Tregs were defined as CD4+ CD25highFoxp3+ CD127-cells. The amounts of Tregs were presented as a median proportion of CD4-positive cells. Two forms of CTLA-4: surface (s) and intracellular (in) were analyzed in different tubes. For Foxp3 and (in)CTLA-4 detection, membrane permeabilization with Transcription Buffer Set (BD, USA) was used. CTLA-4-positive cells were shown as a median proportion of Tregs. The samples were processed by the FACS Canto II flow cytometer (BD, USA).
3
Comprehensive B-cell Immunophenotyping Protocol
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To investigate B‐1 cell populations pre‐ or poststimulation, splenocytes and peritoneal cells were stained with CD19‐V450, CD43‐PE, CD5‐APC, IgM‐PE‐Cy7, and CD125‐FITC. For analysis of B‐1 cell intracellular IgM‐expression after stimulation, cells were initially stained on the surface with CD19‐V450, CD43‐FITC, CD5‐APC, and IgM‐PE. Fixation, permeabilization, and intracellular staining were performed using the Transcription Buffer Set (BD Bioscience) and IgM‐PE‐Cy7 antibody. To analyze splenic GC B cells and TFH cells, splenocytes were stained with B220‐PerCP, CD21‐FITC, CD23‐BV510 and GLY7‐PE, or CD3‐FITC, CD4‐PacificBlue, CXCR5‐APC and GLY7‐PE, respectively. MZ and FO B cells were stained using B220‐PerCP, CD23‐BV510, CD21‐FITC, IgM‐APC‐Cy7, and IgD‐V450. All fluorchrome‐conjugated antibodies were purchased from BD Bioscience or Biolegend. Before surface staining, unspecific FC‐binding sites were blocked by incubation with anti‐CD16/32 (Biolegend) for 15 min. Measurements were performed at the BD FACSCanto II and gating was done using FlowJo Version 10. To ensure correct gating, fluorescence minus one controls were applied.
4
Multiparametric Flow Cytometry Analysis
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Il-4Rα surface expression was detected on lymph node cells by phycoerythrin (PE) anti-CD124 (IL-4Rα, M-1). Cell subpopulations were identified with Alexa Fluor 700, BD Horizon V500, BD Horizon V450, PerCP-Cy5.5, APC, APC-Cy7, Fluoroscein isothiocyanate, PE, PE-Cy7 or biotinylated monoclonal antibodies against CD3, CD4, CD19, Lineage, Gata-3, IL-4, IL-13, IFN-γ, IL-10, SiglecF, T1/ST2, ICOS. Biotin-labeled antibodies were detected by Allophycocyanin or Percpcy5.5. For staining, cells (1x 106) were labeled and washed in PBS, 3%FCS and 0.1% NaN3. Between each step of staining, cells were washed extensively. For intracellular cytokine staining, cells were restimulated with a cocktail of PMA/Ionomycin/Monensin for 4–12 h at 37°C then fixed in 2% PFA, permeabilized and cytokine production was analyzed as previously described [28 (link)]. For intranuclear staining, a commercially available transcription buffer set (BD Bioscience) was used as per the manufacturer’s instructions. All antibodies were from BD Pharmingen (San Diego, CA) except where noted otherwise. Stained cells were then acquired on a LSR Fortessa machine (BD Immunocytometry system, San Jose, CA, USA) and data were analyzed using Flowjo software (Treestar, Ashland, OR, Usa).
5
Flow Cytometric Analysis of T Cell Subsets
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Frozen BAL or PBMC samples were thawed and centrifuged, and the cell pellet was resuspended and washed with phosphate-buffered saline (PBS) (Gibco). Cells were stained with the antibodies listed in Table 2 Dead cells were excluded using Fixed Viability Stain 620 (BD Biosciences, Macquarie Park, NSW), and Fc receptor was blocked with TruStain FcX (BioLegend, San Diego, CA, USA). Intracellular staining for FoxP3, granzyme B, and Ki67 was carried out using the Transcription Buffer Set (BD Biosciences). Flow cytometric experiments were conducted using the BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences) and analyzed using KALUZA flow cytometry analysis software version 2.1 (Beckman Coulter, Brea, CA, USA). The gating strategy for identifying T cell populations is illustrated in Figure 1 for both BAL and PBMC. Cell subsets were quantified as percentages, and absolute counts were calculated per 100 mL of BAL. Table 2 lists the markers included in the study.
6
Comprehensive Immune Profiling of IFNα-Treated PBMCs
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PBMCs collected before and after 1 week of IFNa treatment were thawed and divided into multiple samples that were stained with separate antibody panels for myeloid-derived suppressor cell (MDSC), inhibitory/memory, regulatory T cell and dendritic cell (DC) markers, respectively (online supplementary table 1a ). Dead cells were stained using Yellow ArC-Qdot585 (ThermoFisher, L34959).
Staining was carried out according to our standard protocols,24 (link) washed with Fluorescence Activated Cell Sorting (FACS) buffer, fixed in 1% paraformaldehyde and analyzed using a LSRFortessa X20 (BD Biosciences).
Staining of the regulatory T cell panel was conducted using the Transcription Buffer Set (BD Biosciences) as previously described.25 (link) FACS results were analyzed with BD FACSDiva software (V.8.02).
Staining was carried out according to our standard protocols,24 (link) washed with Fluorescence Activated Cell Sorting (FACS) buffer, fixed in 1% paraformaldehyde and analyzed using a LSRFortessa X20 (BD Biosciences).
Staining of the regulatory T cell panel was conducted using the Transcription Buffer Set (BD Biosciences) as previously described.25 (link) FACS results were analyzed with BD FACSDiva software (V.8.02).
7
Intracellular Flow Cytometry of Zbtb46
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Cells were kept at 4 °C while being stained in PBS with 0.5% BSA and 2 mM EDTA in the presence of CD16/32 Fc block (BD; clone 2.4G2). For intracellular flow cytometry, cells were stained for surface markers, permeabilized, and fixed with the transcription buffer set (BD) following the manufacturer’s instructions. Cells were then stained for intracellular Zbtb46 expression at 4 °C on ice. Cells were analyzed on a FACS Canto II and sorted on a FACS Aria Fusion flow cytometers (BD). Data were analyzed with FlowJo software (Tree Star). Antibodies and other staining materials are described in SI Appendix, Materials and Methods .
8
Intracellular IL-17A Staining in Activated PBMCs
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Total PBMCs were cultured at 37°C, 5% CO 2 (RPMI medium, 10 5 cells per well) for 24 h in the presence of T cell activation and expansion beads (TAE, anti-CD3/CD28/CD2 mAb micro beads; Miltenyi Biotech, Bergisch Gladbach, Germany), providing polyclonal stimulation, or with culture media alone. Four hours prior to cell harvest, 2 μg Brefeldin A (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) was added to each culture. The cells were stained for extracellular markers with monoclonal antibodies (described above), followed by permeabilisation and intracellular staining using Transcription Buffer Set (BD Biosciences; intracellular staining protocol) and anti-human IL-17A antibody (Biolegend). Unstained and fluorescence minus one cells (stained with similar reagents but omitting the anti-IL-17 antibody) were used to confirm positive intracellular staining. Stained sample acquisition and data analysis are described above (ESM Fig. 1) with the addition of gating for IL-17 + cells.
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