For cell cycle analysis, thymocytes were stained with surface markers followed by fixation and permeabilization with eBioscience Transcription Factor Staining Buffer Set (Life Technologies, 00-5523-00). The cells were then stained with anti-Ki-67 (FITC, 16A8, 652409, Biolegend) and DAPI for cell cycle analysis. For BrdU staining, total thymocytes were stained with surface markers to define each subset, followed by intracellular staining using the BrdU staining kit according to the manufacturer’s instructions (BD Biosciences, 559619).
Ebioscience transcription factor staining buffer set
Manufactured by Thermo Fisher Scientific
The EBioscience transcription factor staining buffer set is a laboratory reagent designed to facilitate the detection and analysis of transcription factors in cells. The set includes buffers for cell fixation, permeabilization, and staining, enabling researchers to effectively prepare samples for flow cytometry or microscopy analysis of intracellular transcription factor expression.
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Lab products found in correlation
Brdu staining kit, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Fc receptor blocking agent, by Miltenyi Biotec (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Pe conjugate, by Cell Signaling Technology (1 mentions)
Amnis imagestream mark 2, by Merck Group (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
Siglecf pe, by BD (1 mentions)
Ki67 fitc, by BioLegend (1 mentions)
Cd11c bv421, by BioLegend (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
Cd3 af488, by BioLegend (1 mentions)
Cd4 bv786, by BD (1 mentions)
Cd8a bv650, by BioLegend (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Zombie nir, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
7 protocols using ebioscience transcription factor staining buffer set
1
Cell Cycle Analysis of Thymocytes
2
Thymocyte Cell Cycle Analysis
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For cell cycle analysis, thymocytes were stained with surface markers followed by fixation and permeabilization with eBioscience Transcription Factor Staining Buffer Set (Life Technologies, 00-5523-00). The cells were then stained with anti-Ki-67 (FITC, 16A8, 652409, Biolegend) and DAPI for cell cycle analysis. For BrdU staining, total thymocytes were stained with surface markers to define each subset, followed by intracellular staining using the BrdU staining kit according to the manufacturer's instructions (BD Biosciences, 559619).
3
Comprehensive Immune Profiling of Tumor Samples
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After tumor digestion, the single-cell suspension was rinsed with phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3). After 15 min of incubation with Fc receptor blocking agent (1:25, Miltenyi), the cells were stained with antibodies against CD45, epidermal growth factor receptor (EGFR), CD3, CD4, CD8, CD11c, CD19, CD45RA APC-H7, CD25, lymphocyte-activation gene 3 (LAG-3), PD-1, CD80 and programmed death-ligand 1 (PD-L1) by incubating them for 30 min at 4°C. Details on the fluorochrome-conjugated monoclonal antibodies can be found in the online supplemental methods . For intracellular staining, the eBioscience transcription factor staining buffer set (Invitrogen) was used to fix and permeabilize the cells. Next, the cells were stained with antibodies against forkhead box P3 (FOXP3), glycoprotein A repetitions predominant (GARP) or IgG2b by 30 min incubation at 4°C. A BD LSR Fortessa X20 flow cytometer was used for data acquisition and analyses were performed using FlowJo software V.10.8.1. Detailed descriptions of the flow cytometry analyses are provided in the online supplemental methods .
4
IFN-Induced STAT1 Localization in HeLa Cells
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Hela cells transfected with NC or ASOs were stimulated with IFN (1,000 units/ml) for 30 min and then fixed and permeated with eBioscience transcription factor staining buffer set (Invitrogen) according to the manufacturer's protocol. Cells were resuspended in 100 μl of FACS buffer and incubated with antibody (diluted by 1:50) for at least 30 min at room temperature in dark. DAPI was used to stain cell nuclei for <3 min. Cells were then washed and resuspended in FACS buffer (volume between 20 and 200 μl) in an appropriate cell concentration of 1–2 × 107/ml. Cell samples were loaded and analyzed using Amnis ImageStream MarkII (Merck). Similarity between STAT1 and nuclei staining pattern were calculated. The antibody used in flow imaging cytometry experiment was STAT1 rabbit mAb (PE Conjugate; Cell Signaling Technology).
5
Intracellular Staining for EBF1 Analysis
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For intracellular staining, cells were fixed and permeabilized with eBioscience transcription factor staining buffer set (Thermo Fisher, 00-5523-00). Cells were stained with anti-EBF1 (peptide purified; BioGenes) or normal rabbit IgG (Cell Signaling, 2729). Alexa fluor 488-conjugated anti-rabbit IgG (Thermo Fisher, A-11034) was used to label the primary antibodies. Cells were analyzed using BD LSRII. The data were processed and visualized with FlowJo. CD19 staining was performed as described (Boller et al. 2016 (link)) with APC-coupled anti-CD19 antibody (BD Pharmingen, 550992).
6
Isolation and Analysis of Lung Immune Cells
7
Multiparameter Flow Cytometry Analysis
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Thymocytes and TEC were stained as indicated, using antibodies and reagents listed in Supplementary Table 1 , including dilutions. For intracellular staining, cells were fixed, permeabilized (Cytofix/Cytoperm Kit, BD Biosciences) and labelled except for Foxp3 staining, where the eBioscience transcription factor staining buffer set (ThermoFisher Scientific) was used. Stained samples were acquired on a FACSAria II or BD LSRFortessa flow cytometer with FACSDiva (BD Biosciences v8.01) and the data were analysed using the FlowJo (Treestar Inc. v9.8.3 and v10.7.1) software.
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