Following CD4+ T-cell isolation, cells were stimulated with EBOV at the indicated MOI for 48 h and surface stained for CD25-Alexa.Fluoro.700 clone M-A251 (BioLegend) and CD69-PE/Dazzle clone fn50 (BioLegend). Cells were fixed and permeabilized using the True Nuclear transcription factor kit (BioLegend) and stained for intracellular Ki-67-BVLT.421 clone B56 (BD Biosciences), IL-2–APC clone MQ1-17H12 (BD Biosciences), TNF-α–FITC clone Mab11 (BD Biosciences), and IFN-γ–PE clone B27 (BD Biosciences). Samples were then analyzed using a FACS Fortessa instrument (BD Biosciences). Cytokines and chemokines were detected and quantitated from supernatants by Eve Technologies (Calgary, Alberta, CA). Heat maps were generated using GENE-E (Broad Institute; http://www.broadinstitute.org/cancer/software/GENE-E/index.html ).
True nuclear transcription factor kit
Manufactured by BioLegend
Sourced in United States
The True Nuclear Transcription Factor Kit is a comprehensive solution for the analysis of transcription factor activation in nuclear extracts. It provides a sensitive and specific method for the detection and quantification of transcription factors in a 96-well format.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α fitc clone mab11, by BD (1 mentions)
Ifn γ pe clone b27, by BD (1 mentions)
Facs fortessa, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Zombie nir fixable viability dye, by BioLegend (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
6 protocols using true nuclear transcription factor kit
1
Characterization of EBOV-Induced T-Cell Activation
2
Phenotypic Analysis of Human and Mouse T Cells
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For each experimental condition, cells from human PBMCs, mouse peripheral blood, and mouse splenocytes were isolated and labeled with antibodies at 4°C for 45 min in the dark as follows. Anti-human antibodies: CD3 (300412, clone UCHT1, 1:300), CD4 (17-0049-73, clone RPA-T4, 1:300), CD8 (301008, clone RPA-T8, 1:300), CD25 (302606, clone BC96, 1:200), IL-2 (500306, clone MQ1-17H12, 1:300), IFN-γ (502523, clone 4S.B3, 1:300), CD62L (304813, clone DREG-56, 1:300) CD45RA (304110, clone HI100, 1:300); Anti-human/mouse antibody Granzyme B (515403, clone GB11, 1:300); Anti-mouse antibodies: CD3 (100321, clone 145-2C11, 1:300), CD4 (100407, clone GK1.5, 1:200), CD8 (100713, clone 53-6.7, 1:300), and CD25 (10211, clone PC61, 1:200). All antibodies were from Biolegend. All samples were pre-incubated with TruStain fcX (101320, clone 93, 1:100, Biolegend) to block the Fc receptors. Intracellular staining was carried out per the manufacturer’s instruction described in the True Nuclear Transcription Factor Kit (424401, Biolegend). Samples were acquired on the FACS Canto (BD Biosciences) and analyzed using FlowJo version 10.1 (Becton, Dickinson & Company). Dead cells were excluded from the analysis based on the forward and side scatter characteristics.
3
Immunophenotyping of single-cell suspensions
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Single‐cell suspension was incubated for 30 min at 4°C with Fc block (anti‐mouse CD16/32 Ab) to prevent non‐specific binding of Abs. Next, cell‐surface staining was done with fluorochrome‐tagged monoclonal antibodies for another 30 min at 4°C as per the experiment. For intracellular staining, cell surface stained cells were fixed and permeabilized using True‐nuclear™ transcription factor kit‐Biolegend (San Diego, CA) and stained for intracellular targets in accordance with the manufacturer's protocol. The data was obtained with BD FACSVerse and analyzed through BD FlowJo software (San Jose, CA).
4
Multiparameter Flow Cytometry of Immune Cells
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Single-cell suspensions from all samples were prepared by straining through a 70 µm filter. Cells were pre-incubated for 5 minutes at room temperature with CD16/CD32 masking antibody (clone 2.4G2, Tonbo Bioscience, San Diego, CA, catalog no. 70-0161-U500) to prevent non-specific antibody binding. Cells were stained with Zombie-NIR™ fixable viability dye (Biolegend, catalog no. 423106) to differentiate live/dead cells. Then, cells were stained in 1x DPBS/2% FCS/5 mM EDTA with the appropriate surface antibodies (Table S1 Table S1
5
Multicolor Flow Cytometry Staining
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The single-cell suspension was stained with different antibodies against cell surface markers and isotype controls in flow cytometry buffer (PBS supplemented with 5 % FBS, each 1:100). A list of the antibodies used for this study can be found on Table 1 . The cells were stained at 4 °C for 25 min in the dark. Afterwards, either Zombie NIR (1:50) or propidium iodine (1:100) was added and incubated for a further 5 min at 4 °C. Then, the cells were spun down at 8050 RCF× g at 4 °C for 7 min and washed twice with flow cytometry buffer and centrifuged. The cells were then resuspended and transferred to FACS tubes. For intracellular staining of FOXP3, the cells were transferred into 15 mL tubes and treated with True-Nuclear™ Transcription Factor kit from BioLegend® (Cat.: 424401, BioLegend, San Diego, CA, USA) and stained with either an antibody against FOXP3 or an isotype control following the manufacturer’s instructions.
6
Multi-Tissue Single-Cell Staining
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Single-cell suspensions were prepared from various tissues. Surface staining was performed after FcgRII/III blocking for 5 minutes at 4 C, and cells were surface stained for 30 minutes at 4 C. For staining of transcription factors, cells were stained using the True-Nuclear Transcription Factor kit (Biolegend). In all experiments, dead cells were excluded using Fixable Viability Dye (eBioscience).
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