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Transcription factor buffer set

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The Transcription Factor Buffer Set is a collection of buffers designed to facilitate the extraction and analysis of transcription factors from cell samples. The set includes reagents for nuclear protein extraction, DNA-protein binding, and electrophoretic mobility shift assays (EMSA). The core function of this product is to provide a standardized and optimized solution for the isolation and study of transcription factors, which play a crucial role in gene expression regulation.

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Lab products found in correlation

Countbright beads, by Thermo Fisher Scientific (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Lsrfortessa, by BD (1 mentions) Golgistop, by BD (1 mentions) Cell viability dye, by Thermo Fisher Scientific (1 mentions) Apc conjugated streptavidin, by BioLegend (1 mentions) Facscanto 2, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions) Cd4 fitc, by BioLegend (1 mentions) Cd69 pe cy7, by BioLegend (1 mentions) Cd8 alexa fluor 700, by BD (1 mentions) Mhc 1 pe, by Thermo Fisher Scientific (1 mentions) Cd44 bv650, by BioLegend (1 mentions) Cd11c apc, by Thermo Fisher Scientific (1 mentions) Pd1 pecf594, by BD (1 mentions) Cd3 percp cy5, by BD (1 mentions) Cd45 bv510, by BD (1 mentions) Pd l1 pe, by BD (1 mentions)

5 protocols using transcription factor buffer set

1

Quantifying Immune Cell Subsets

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Absolute B-cell, T-cell, and natural killer (NK) cell numbers in peripheral blood were determined by using flow cytometry with CountBright beads (Thermo Fisher, Waltham, Mass) with an Fc-blocking reagent after incubation at 4°C and gating on live CD45+ cells. ILC populations were identified as CD3−CD14−CD11c−CD19−CD117+CD127+ cells (ILCs); CD3−CD14− CD19−CD117−CD127−CD56+ cells were identified as conventional NK cells. For analysis of intracellular transcription factors, PBMCs were permeabilized, fixed with Transcription Factor Buffer Set (eBioscience, San Diego, Calif) and labeled with mAbs to human retinoic acid–related orphan receptor (ROR) γt or T-box transcription factor (T-bet; antibodies are shown Table E1 in this article’s Online Repository at www.jacionline.org). For intracellular cytokine staining, PBMCs were cultured for 4 hours in RPMI medium with GolgiStop (BD PharMingen, San Jose, Calif). Those cells were stained with antibodies to specific surface molecules, fixed, and permeabilized (Cytofix/Cytoperm Kit, BD PharMingen). Cells were examined by using LSR Fortessa (BD) and analyzed with FlowJo software (Tree Star, Ashland, Ore).
Cols M., Rahman A., Maglione P.J., Garcia-Carmona Y., Simchoni N., Ko H.B., Radigan L., Cerutti A., Blankenship D., Pascual V, & Cunningham-Rundles C. (2015). Expansion of inflammatory innate lymphoid cells in patients with common variable immune deficiency. The Journal of allergy and clinical immunology, 137(4), 1206-1215.e6.
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2

Single-cell Analysis of Insulin-Binding B Cells

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Single-cell suspensions were treated with FcR-blocking media containing 2.4G2 antibodies. The cells were then stained with fluorescent antibodies on ice for 25 min followed by incubation with cell viability dye (eBioscience). For detection of insulin-binding B cells, cells were incubated with 50 ng/ml biotinylated insulin (Eagle Biosciences) with or without inhibition with 500 ng/ml unconjugated insulin for 30 min on ice followed by staining with APC-conjugated streptavidin (BioLegend) for 15 min. The FoxP3 staining was performed using a transcription factor buffer set (eBioscience). The flow cytometry samples were collected using a FACSCanto II (BD), and data were analyzed using FlowJo software (Tree Star). Cell sorting was performed using a FACSAria II (BD).
Wan X., Thomas J.W, & Unanue E.R. (2016). Class-switched anti-insulin antibodies originate from unconventional antigen presentation in multiple lymphoid sites. The Journal of Experimental Medicine, 213(6), 967-978.
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3

FoxP3 Induction in OVA-specific T Cells

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We followed the induction of FoxP3 expression in OVA-specific T cells injected in 1-day-old mice following a previously published protocol, 9 which is described in the Online Repository. One week after the last OVA and/or D pteronyssinus administration to mothers, mesenteric lymph nodes were harvested and cell suspension analyzed by flow cytometry. Injected DO-11.10 CD4 T cells were identified using anti-CD3, anti-CD4, and KJ1.26 mAb (eBioscience). OVA-specific CD4 T cells were analyzed for FoxP3 expression after cell fixation and permeabilization using transcription factor buffer set (eBioscience).
Rekima A., Bonnart C., Macchiaverni P., Metcalfe J., Tulic M.K., Halloin N., Rekima S., Genuneit J., Zanelli S., Medeiros S., Palmer D.J., Prescott S, & Verhasselt V. (2020). A role for early oral exposure to house dust mite allergens through breast milk in IgE-mediated food allergy susceptibility. The Journal of allergy and clinical immunology, 145(5).
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4

Visualization of Virus-Specific T Cells

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Biotinylated MHC class I H2-Db and class II I-Ab monomers specific for the RHV NS968 and NS31265 epitopes were obtained from the NIH Tetramer Core Facility and tetramerized with streptavidin-PE (Prozyme). For direct visualization of virus-specific T cell populations, liver-infiltrating leukocytes or splenocytes were stained for 60 min at 4°C (1:500) for Class-I and 90 min at 37°C for Class-II tetramer, followed by labeling with antibodies for surface markers. Subsequently, cells were stained LIVE/DEAD Fixable Near-IR Dead Cell Stain kit, fixed, and permeabilized by transcription factor buffer set (eBiosciences) before staining for transcription factors.
Dravid P., Murthy S., Attia Z., Cassady C., Chandra R., Trivedi S., Vyas A., Gridley J., Holland B., Kumari A., Grakoui A., Cullen J.M., Walker C.M., Sharma H, & Kapoor A. (2023). Phenotype and fate of liver-resident CD8 T cells during acute and chronic hepacivirus infection. PLOS Pathogens, 19(10), e1011697.
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5

Multiparametric Analysis of Tumor-Infiltrating Immune Cells

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Tumors tissues were harvested, and single-cell homogenates were prepared by using a mouse tumor dissociation kit (Miltenyi Biotec) and passed through a 70-mm strainer before the red blood cells were lysed. Surface markers were stained with the following antibodies at 4 °C for 30 min: CD45-BV510 (563891, BD), CD3-PerCP-Cy5.5 (551163, BD), CD4-FITC (100406, BioLegend), CD8-Alexa Fluor 700 (557959, BD), CD69-PE/Cy7 (104512, BioLegend), CD44-BV650 (103049, BioLegend), PD-1-PE-CF594 (562523, BD) and PD-L1-PE (558091, BD). For forkhead box P3 (FOXP3) staining, a transcription factor buffer set (00-5523-00, eBioscience) was used.
For DCs staining, following antibodies were used: CD11c-APC (17-0114-82, eBioscience), MHC I-PE (12-5958-82, eBioscience) and CD86-eFluor 450 (48-0862-82, eBioscience).
Liu Y., Cai J., Liu W., Lin Y., Guo L., Liu X., Qin Z., Xu C., Zhang Y., Su X., Deng K., Yan G, & Liang J. (2020). Intravenous injection of the oncolytic virus M1 awakens antitumor T cells and overcomes resistance to checkpoint blockade. Cell Death & Disease, 11(12), 1062.
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