Pfi 3

Manufactured by Selleck Chemicals

Sourced in China

The PFI-3 is a laboratory equipment designed for precision fluid injection. It is a versatile device capable of accurately delivering small volumes of liquids in a controlled manner. The core function of the PFI-3 is to provide precise and repeatable fluid dispensing for various laboratory applications.

Automatically generated - may contain errors

Lab products found in correlation

Angiotensin 2, by Merck Group (2 mentions) Luciferase reporter assay system, by Promega (2 mentions) Ea hy926, by American Type Culture Collection (2 mentions) Small interfering rna, by Horizon Discovery (2 mentions) Triptolide, by Selleck Chemicals (1 mentions) α amanitin, by Merck Group (1 mentions) Hmvec, by Lonza (1 mentions) Tnf α, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Raw264, by American Type Culture Collection (1 mentions) 3 kda mw cut off filter units, by Merck Group (1 mentions) Bromosporine, by Merck Group (1 mentions) M0896, by Merck Group (1 mentions) Mocetinostat, by Selleck Chemicals (1 mentions) A3672, by ITW Reagents (1 mentions) Mm 102, by Selleck Chemicals (1 mentions) Ex527, by Selleck Chemicals (1 mentions) Unc0631, by Selleck Chemicals (1 mentions) D5942, by Merck Group (1 mentions)

6 protocols using pfi 3

Small Molecule Modulation of Embryonic Development

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α-Amanitin was purchased from Sigma (A2263-1 mg) and dissolved in 1 ml ddH₂O. For injection, a 1:20 solution was freshly prepared in ddH₂O, and 2 nl (0.2 ng) was injected into embryos at one-cell stage. Triptolide (PG490- 1 mg, Selleckchem) was diluted in dimethyl sulfoxide (DMSO) to obtain a 2.5 mM stock. For drug treatment, the stock was further diluted to 10 mM in embryo medium. Manually dechorionated embryos were reared in the Triptolide solution from the 128-cell stage until 4.3 hpf. PFI-3 (catalog no. S7315-5 mg, Selleckchem) was dissolved in DMSO to a 5 mM stock solution. For drug treatment, dilutions of PFI-3 stock in ddH₂O (1–100 mM) were freshly prepared, thoroughly vortexed, and 1–2 nl was injected into one-cell stage embryos.

Laue K., Rajshekar S., Courtney A.J., Lewis Z.A, & Goll M.G. (2019). The maternal to zygotic transition regulates genome-wide heterochromatin establishment in the zebrafish embryo. Nature Communications, 10, 1551.

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Cellular Signaling in Endothelial Cell Regulation

Cited 1 time

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Immortalized human endothelial cells (EAhy926, ATCC) and HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone). Human primary microvascular endothelial cells (HMVEC) were purchased from Lonza and maintained in EGM-2 media with supplements supplied by the vendor; three different batches of primary cells were used in this study as previously described¹⁸. Primary murine cardiac microvascular endothelial cells were isolated as previously described¹⁹. Angiotensin II was purchased from Sigma. SNAI2/SLUG promoter-luciferase constructs^{20 (link)}, COL1A2 promoter-luciferase constructs^{21 (link)}, BRG1 expression constructs²², SLUG expression constructs^{23 (link)}, Sp1 expression constructs^{24 (link)}, and SRF expression constructs^{25 (link)} have been previously described. PFI-3 was purchased from Selleck. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described^{26 (link)}.

Li Z., Kong X., Zhang Y., Zhang Y., Yu L., Guo J, & Xu Y. (2020). Dual roles of chromatin remodeling protein BRG1 in angiotensin II-induced endothelial–mesenchymal transition. Cell Death & Disease, 11(7), 549.

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TNF-α-induced Endothelial Cell Activation

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Human endothelial cell line Ea.hy926 was cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator. Small interfering RNAs were purchased from Dharmacon. Transient transfection was performed with Lipofectamine 2000 (Invitrogen, United States, 11668019). Cells were harvested 48 h after transfection. TNF-α was purchased from Peprotech (United States, 300-01A-50). PFI-3 was purchased from Selleck (Shanghai, China, S7315). Ea.hy926 was seeded at 1 × 10⁵ cells/p35 culture dish and starved in serum-free DMEM overnight. TNF-α (10 μg/L) was added the next day for another 12 or 24 h. In certain experiments, PFI-3 (2 μM) was added together with TNF-α.

Zhang Y., Wang H., Song M., Xu T., Chen X., Li T, & Wu T. (2020). Brahma-Related Gene 1 Deficiency in Endothelial Cells Ameliorates Vascular Inflammatory Responses in Mice. Frontiers in Cell and Developmental Biology, 8, 578790.

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Endothelial Cell Characterization Protocol

Cited 2 times

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Immortalized human endothelial cells (EAhy926, ATCC), mouse macrophage-like cells (RAW264.7, ATCC), and HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone). Angiotensin II was purchased from Sigma. MRP8 promoter-luciferase constructs (Grebhardt et al., 2012 (link)) and BRG1 expression constructs (Li et al., 2019a (link)) have been previously described. Small interfering RNAs were purchased from Dharmacon. PFI-3 was purchased from Selleck. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described (Li et al., 2019c (link)). For conditioned media (CM) collection, the cells were switched to and incubated with serum-free media overnight. The next day, the media were collected, centrifuged at 4000 × g for 30 min at 4°C using 3-kDa MW cut-off filter units (Millipore) and sterilized through a 0.4-μm filter.

Li Z., Zhang Y., Zhang Y., Yu L., Xiao B., Li T., Kong X, & Xu Y. (2020). BRG1 Stimulates Endothelial Derived Alarmin MRP8 to Promote Macrophage Infiltration in an Animal Model of Cardiac Hypertrophy. Frontiers in Cell and Developmental Biology, 8, 569.

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SMARCA4 Bromodomain Inhibition Assay

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The SMARCA4 bromodomain activity inhibitor, PFI-3 (S7315), was purchased from SelleckChem. Rh30 cells were treated with PFI-3 at a concentration of 10 μM. PFI-3 inhibits 95% of the SMARCA4 bromodomain activity at 10 μM based on TR-FRET assay carried out by a commercial vendor, BPS bioscience. Briefly, the assay was performed by TR-FRET technology using recombinant bromodomain and BET Ligand. The TR-FRET signal from the assay is correlated with the amount of Ligand binding to the bromodomain. Binding experiments were performed in duplicate at each concentration. The TR-FRET data were analyzed using the computer software, GraphPad Prism (GraphPad Software Inc.). In the absence of the compound in wells containing BET ligand, the TR-FRET signal (Ft) in each data set was defined as 100% activity. Select wells where control inhibitors were more than 100-fold the IC₅₀ were used to define the TR-FRET signal (Fb) as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity = [(F-Fb)/(Ft - Fb)]×100, where F= the TR-FRET signal in the presence of the compound. The percent inhibition was calculated according to the following equation: % inhibition = 100 - % activity.

Bharathy N., Berlow N.E., Wang E., Abraham J., Settelmeyer T.P., Hooper J.E., Svalina M.N., Ishikawa Y., Zientek K., Bajwa Z., Goros M.W., Hernandez B.S., Wolff J.E., Rudek M.A., Xu L., Anders N.M., Pal R., Harrold A.P., Davies A.M., Ashok A., Bushby D., Mancini M., Noakes C., Goodwin N.C., Ordentlich P., Keck J., Hawkins D.S., Rudzinski E.R., Chatterjee B., Bächinger H.P., Barr F.G., Liddle J., Garcia B.A., Mansoor A., Perkins T.J., Vakoc C.R., Michalek J.E, & Keller C. (2018). The HDAC3-SMARCA4-miR27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma. Science signaling, 11(557), eaau7632.

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Screening for Epigenetic Modulators

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Supplementary Figure 1 lists all small molecule compounds used in the screens. Selected epigenetic small molecule compounds were obtained from the Collaborative Research Centre 992 Medical Epigenetics (CRC992 MEDEP; Laboratory of Manfred Jung) at University of Freiburg, and from the Structural Genomics Consortium (SGC). Additionally, the following chemical compounds were obtained commercially: Bromosporine (Sigma #SML0992), Ex-527 (Selleckchem #S1541), MM-102 (Selleckchem #S7265), Mocetinostat (Selleckchem #S1122), OICR9429 (Tocris # 5267), PFI-3 (Selleckchem #S7315), UNC0631 (Selleckchem, S7610). All small molecule compounds were dissolved in DMSO (99.5%, PanReac AppliChem #A3672) at 10 mM concentration. For experiments, the 10 mM stock was diluted with E3 medium to obtain the desired working stock concentration of 60 µM containing 1% DMSO, which was used to prepare the dilution series. The γ-secretase inhibitor DAPT (increased DA neurogenesis; (Mahler et al., 2010 (link)); Sigma, #D5942) and the prodrug neurotoxin MPTP (neurotoxic to DA neurons; (Sallinen et al., 2009 (link)); Sigma #M0896) were used as positive controls for drug delivery into embryos and dopaminergic neurons.

Westphal M., Sant P., Hauser A.T., Jung M, & Driever W. (2020). Chemical Genetics Screen Identifies Epigenetic Mechanisms Involved in Dopaminergic and Noradrenergic Neurogenesis in Zebrafish. Frontiers in Genetics, 11, 80.

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