THP-1 cells (ATCC® TIB-202™) were used as phagocytic cells. For this, 1 µg of biotinylated S1 protein (Genscripts) was used to saturate the binding sites on 1 µl of FluoroSphere neutravidin beads (Thermo Fisher) overnight at 4°C. Excess protein was removed by washing the pelleted beads. The protein-coated beads were incubated with 40 µl heated-inactivated plasma diluted in complete RPMI (1:40) for 15 minutes at room temperature. Then, 5x104 THP-1 cells suspended in 50 µl complete RPMI were added to the complex and incubated for 16 hours at 37°C, 5% CO2. After incubation, the cells were washed with 1xPBS and fixed using buffer in True-Nuclear Transcription Factor Staining kit (Biolegend). After fixing, the cells were washed and resuspended in 1xPBS. The samples were analyzed using FACS Canto II, BD Biosciences. Phagocytosis activity was scored by the integrate mean fluorescence intensity (iMFI) value (% positive fluorescence THP-1 cells x MFI of the positive fluorescence THP-1 cells).
True nuclear transcription factor staining kit
Manufactured by BioLegend
The True-Nuclear Transcription Factor Staining kit is a laboratory equipment product designed for the detection and analysis of transcription factors within the cell nucleus. It provides a reliable method for staining and quantifying nuclear transcription factors.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (4 mentions)
Zombie aqua viability dye, by BioLegend (2 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
Anti igm pe, by BioLegend (1 mentions)
Anti igg alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Negative magnetic selection, by Miltenyi Biotec (1 mentions)
Anti cd107a, by BioLegend (1 mentions)
Monensin, by BioLegend (1 mentions)
Specific antibodies, by BioLegend (1 mentions)
Antirat cd32 antibody, by BD (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
7 protocols using true nuclear transcription factor staining kit
1
SARS-CoV-2 S1 Protein Phagocytosis Assay
2
Quantifying SARS-CoV-2 Antibody Neutralization
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The assay used spike-expressing Raji cells as target cells, pooled serum (4 healthy donors) as complement source and heated-inactivated patient plasma as antibody source. In short, 50 µl of heated-inactivated plasma (1:50) were incubated with Raji-spike cells for 30 minutes at 37°C, 5% CO2. Afterward, 50 µl of complete RPMI containing 15% of pooled serum was added into the cells and incubated at 37°C, 5% CO2 for 14 hours. The cells were washed with PBS and stained with Zombie Aqua viability dye (BioLegend) for 20 minutes on ice and then stained anti-APC C3/C3b/iC3b antibody (Cedarlane) for 30 minutes on ice. The cells were fixed with fixation buffer in True-Nuclear Transcription Factor Staining kit (Biolegend) for 20 minutes on ice. After fixing, the cells were washed and resuspended in 1xPBS. The samples were acquired using FACS Canto II, BD Biosciences. The results were reported as percentage of cell death and MFI of C3 deposition on the cells.
3
Quantitative NCC Transcriptomics and Proliferation
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E13.5 anterior facial regions were dissected and dissociated in 0.25% trypsin and 0.7 mg/ml DNase I (in Hanks’ Balanced Salt Solution) for 10 min at 37°C before pipetting and 12% fetal bovine serum (FBS) neutralization. Cells were stained with RUNX2 (1:800, Cell Signaling Technology, 12556S) using the True-Nuclear transcription factor staining kit (BioLegend, 424401) according to the manufacturer's directions. In a similar fashion, anterior E13.5 NCCs were dissociated and flow sorted (University of North Carolina Flow Cytometry Core Facility) based on Tomato reporter fluorescence to prepare RNA for sequencing. Cell trace far red proliferation and tracking dye (Thermo Fisher Scientific, C34572) labeled cells in culture as directed and was quantified by flow cytometry at indicated time points.
4
Transcription Factor Analysis by Flow Cytometry
5
SARS-CoV-2 Antibody Detection by S-Flow Assay
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The S-Flow assay was performed as previously described (39 (link)). Briefly, plasma samples were diluted (1:200) in 1xPBS with 2mM EDTA and 0.5% BSA (PBS/BSA/EDTA) and incubated with 293T-spike cells (80000 cells/100µl) for 30 minutes on ice. The cells were washed with PBS/BSA/EDTA and stained either with anti-IgM PE (dilution 1:100, Biolegend) and anti-IgG Alexa Fluor™ 647 (dilution 1:600, Thermo Fisher) or anti-IgA Alexa Fluor 647 (dilution 1:800, Jakson ImmunoResearch) for 30 minutes on ice. The cells were washed with 1xPBS and fixed using buffer of the True-Nuclear Transcription Factor Staining kit (Biolegend). After fixing, the cells were washed and resuspended in 1xPBS. The results were acquired using FACS Canto II, BD Biosciences. The gating strategy for anti-IgM, anti-IgG or anti-IgA positive cells was based on the 293T control cells incubated with negative SARS-CoV-2 reference plasma. The data were reported as percentage of positive cells for anti-IgM, anti-IgG or anti-IgA. The NIBSC Research Reagent (20/130) and panel (20/118) (WHO Solidarity II) was utilized to set the cutoff for positivity based on the background staining of the negative SARS-CoV-2 plasma and calculated following formula: cut-off= % positive cells + 2x standard deviation.
6
NK Cell-Mediated Cytotoxicity Assay
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The assay used 293T-spike cells as a target cell and purified NK cells from healthy donor PMBCs as effector cells. First, 293T-spike cells were incubated with heated-inactivated patient plasma diluted in complete DMEM medium (1:50) at 37°C, 5% CO2 for 30 minutes. The NK cells were enriched by magnetic negative selection (Miltenyi) according to manufactor’s instruction. The 293T-spike cells were washed five times with complete RPMI medium. The NK cells were mixed with 293T-spike cells at a ratio 1:1 at final volume of 100 µl complete RPMI. Anti-CD107a and Monensin (Biolegend) 1:1000 dilution were added to the suspension and incubated at 37°C, 5% CO2 for 6 hours. The cells were washed with 1xPBS and stained with Zombie Aqua viability dye (BioLegend) for 20 minutes on ice. Then the cells were stained with anti-CD3 and anti-CD56 for 30 minutes on ice. The cells were washed and fixed/permeabilized using True-Nuclear Transcription Factor Staining kit (Biolegend) for 20 minutes on ice. After staining, the cells were washed and resuspended in 1xPBS. The samples were acquired using FACS Canto II, BD Biosciences.
7
Lymphocyte Immunophenotyping by Flow Cytometry
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Lymphocytes were stained according to standard protocols. Briefly, for staining surface markers, cells were resuspended in fluorescence‐activated cell‐sorting (FACS) buffer at a concentration of 2×106 cells/mL. Nonspecific antibody binding was blocked with anti–rat CD32 antibody (BD Biosciences, Franklin Lakes, NJ) followed by staining with specific antibodies (1:1000, all from BioLegend, San Diego, CA) for 60 minutes on ice in the dark. Antibodies used for flow cytometry included FITC‐CD3, APC‐CD3, PECy7‐CD4, PE‐CD25, FITC‐CD8, APC‐CD45RA, and Alexa Fluor 647‐Foxp3. Intracellular Foxp3 staining was performed after cell surface staining using the True Nuclear transcription factor staining kit (BioLegend) according to the manufacturer's protocol. Stained cells were washed twice and resuspended in FACS buffer and analyzed or sorted on a BD FACSAria II flow cytometer (BD Biosciences).
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