P3000 reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The P3000 reagent is a laboratory product manufactured by Thermo Fisher Scientific. It is used in various scientific applications that require a specific reagent. The core function of the P3000 reagent is to enable researchers and scientists to perform their experiments and analyses effectively. However, a detailed description of the reagent's specific properties and intended use cannot be provided while maintaining an unbiased and factual approach.

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P3000 (95 citations) P3000 Enhancer Reagent (16 citations) Lipofectamine 3000/P3000 reagent (3 citations) Lipofectamine P3000 reagent (2 citations) Lipofectamine 3000 with P3000 reagent (2 citations)

94 protocols using p3000 reagent

Efficient Gene Transfection with NHEJ Inhibitors

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To improve editing efficiency, all cells were treated with NHEJ inhibitors NU7441 (2 µM; Tocris Bioscience) and SCR7 (1 µM; Xcess Biosciences) 4 h before transfection and for 48 h after transfection. HEK293, HeLa and HepG2 cells were seeded in 24-well dishes to reach 60% confluency on the day of transfection. Cells were transfected using Lipofectamine 3000 and P3000 reagent (ThermoFisher Scientific) according to manufacturer's instructions. Plasmids were introduced into HCT116 and MDCK cells using nucleofection with the 384-well HT Nucleofector and the SE cell line kit (Lonza) as per manufacturer's instructions. The cells were nucleofected using the EN-113 program for HCT116 cells, and the CA-152 program for MDCK cells, and the amount of repair template and plasmid were adjusted per cell type.
For transient transgene expression, vectors (pGG-mNG-Anillin, pGG-mNG-Ect2, pGG-mScar-Ect2(C-term) and GFP-RhoA) were transfected into HeLa cells plated on coverslips and grown to 60% confluency, using Lipofectamine 3000 and P3000 reagent (ThermoFisher Scientific) as per manufacturer's instructions.

Husser M.C., Ozugergin I., Resta T., Martin V.J, & Piekny A.J. (2022). Cytokinetic diversity in mammalian cells is revealed by the characterization of endogenous anillin, Ect2 and RhoA. Open Biology, 12(11), 220247.

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PINE-TREE and RoT Transfection in HEK293T

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For PINE-TREE-based transfection, HEK293T cells were transfected in 24-well tissue culture plates 24 h after seeding, when cells were around 40% confluent. 300 ng PE (pEF1α-PE2), 100 ng pegRNA expression plasmid (PE-pegRNA(DT)-nsgRNA(DT)), and 100 ng pEF1α-BFP were transfected per well using 1 μL Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific) and 0.75 μL P3000 reagent (Thermo Fisher Scientific). For RoT transfection, 300 ng PE (pEF1α-PE2), 100 ng pegRNA expression plasmid (PE-pegRNA(DT)-nsgRNA(DT)), and 10 ng pEF1α-GFP were transfected per well using 1 μL Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) and 0.75 μL P3000 reagent (Thermo Fisher Scientific). The medium was changed 24 h post-transfection. Cells were dissociated using Accutase 72 h post transfection and passed through a 0.40-μm filter before sorting.

Frisch C., Kostes W.W., Galyon B., Whitman B., Tekel S.J., Standage-Beier K., Srinivasan G., Wang X, & Brafman D.A. (2023). PINE-TREE enables highly efficient genetic modification of human cell lines. Molecular Therapy. Nucleic Acids, 33, 483-492.

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GFP-K-Ras, GFP-tH, or mCherry-CAAX Transfection in U2OS Cells

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For GFP-K-Ras^G12V, GFP-tH, or mCherry-CAAX transfection in U2OS cells, 1 μg plasmid was mixed with 1.5 μl Lipofectamine 3000 (Life Technologies) and 2 μl P3000 reagent (Life Technologies) in Opti-MEM (Gibco) and incubated for 20 min at room temperature. The medium of the cells to be transfected was then changed to Opti-MEM and the transfection mixture was added. Next, the cells were incubated at 37°C in Opti-MEM. After 4 h, the medium was changed back to regular culture medium and the cells were allowed to recover for additional 4 h before imaging.

Liang H., Mu H., Jean-Francois F., Lakshman B., Sarkar-Banerjee S., Zhuang Y., Zeng Y., Gao W., Zaske A.M., Nissley D.V., Gorfe A.A., Zhao W, & Zhou Y. (2019). Membrane curvature sensing of the lipid-anchored K-Ras small GTPase. Life Science Alliance, 2(4), e201900343.

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UGCG Vector Transfection and Puromycin Selection

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2.5 × 10⁴ Lovo cells were seeded in a 24-well plate for 24 h and transfected with a mixture of UGCG vector DNA or empty vector as control, P3000 reagent and lipofectamine 3000 (Life Technologies, Darmstadt, Germany) according to the manufacturer’s instructions. The selection of transfected cells with 0.5, 1, and 2 µg puromycin/mL culture medium was started 24 h later. The medium was changed one day later and the cells were cultivated for another 3 days in the presence of puromycin. Cells were expanded thereafter in medium without puromycin. After confluence, cells were split to 6-well plates and then to 10 cm dishes. One aliquot was frozen, and one other analyzed for GSL-depletion as described.

Jennemann R., Volz M., Bestvater F., Schmidt C., Richter K., Kaden S., Müthing J., Gröne H.J, & Sandhoff R. (2021). Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo. International Journal of Molecular Sciences, 22(19), 10539.

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miRNA Transfection in hiPSCs

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Transient transfection of hiPSCs with microRNA mimics or inhibitors (Qiagen) was performed using Lipofectamin 3000 without P3000 reagent (Life Technologies, Carlsbad, CA) according to manufacturer’s instructions. Transfections in 24‐well plates were performed using 25 pmol miRNA inhibitor with 12.5 µL Lipofectamin 3000 and 2.5 pmol miRNA mimic with 1.25 µL Lipofectamin 3000. Transfections in 48‐well plates were performed using 10 pmol miRNA inhibitor with 5 µL Lipofectamin 3000 and 1 pmol miRNA mimic with 0.5 µL Lipofectamin 3000. Cells were harvested 48 hours after transfection.

Wiedmann F., Kraft M., Kallenberger S., Büscher A., Paasche A., Blochberger P.L., Seeger T., Jávorszky N., Warnecke G., Arif R., Kremer J., Karck M., Frey N, & Schmidt C. (2022). MicroRNAs Regulate TASK‐1 and Are Linked to Myocardial Dilatation in Atrial Fibrillation. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 11(7), e023472.

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Rspo2 Gene Expression Modulation

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According to the manufacturer's protocol, 2 µl of small interfering (si)RNA (50 nM) or 2 µg plasmid were mixed with 5 µl P3000 reagent (Life Technologies, Grand Island, NY, USA) in 100 µl of opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) medium. Separately, 3.75 µl of lipo 3000 (Life Technologies) was added to another 100 µl of opti-MEM medium. After 5 min of incubation at room temperature, the opti-MEM medium was mixed and added to 1×10⁶ cells in a complete medium for 24 h at 37°C. The siRNA knockdown or plasmid overexpression efficiencies were analyzed by detecting the mRNA levels of Rspo2.

Yan H., Wang S., Li Z., Sun Z., Zan J., Zhao W., Pan Y., Wang Z., Wu M, & Zhu J. (2016). Rspo2 suppresses CD36-mediated apoptosis in oxidized low density lipoprotein-induced macrophages. Molecular Medicine Reports, 14(4), 2945-2952.

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Cell Transfection in 12-well Plates

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SNU398 cells were seeded at a density of 75,000 cells per well in 12-well plates 24 hours before transfection. SNU387 and SNU182 cells were seeded at higher density of 100,000 cells per well in 12-well plates. Plasmid (500 ng) was added to 3 μl of P3000 reagent (Life Technologies) in 75 μl of Opti-MEM before mixing with 2 μl of Lipofectamine 3000 (Life Technologies). The reagent mixture was incubated at room temperature for 10 min before adding to each well.

Kwon J., Liu Y.V., Gao C., Bassal M.A., Jones A.I., Yang J., Chen Z., Li Y., Yang H., Chen L., Di Ruscio A., Tay Y., Chai L, & Tenen D.G. (2021). Pseudogene-mediated DNA demethylation leads to oncogene activation. Science Advances, 7(40), eabg1695.

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Transfection of AgMM Cells with eGFP Plasmid

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The AgMM cells were seeded into a 24-well plate 2–3 days prior to transfection, and transfection experiments were performed when their confluency reached about 80%. The cells were transfected with 1 μl of Lipofectamine 3000 Reagent (Life Technologies), 1 μl of P3000 Reagent and 0.3 μg of a plasmid DNA per well, according to a manufacturer’s protocol. The plasmid contained the eGFP open reading frame under the control of the A. gambiae polyubiquitin promoter, allowing evaluation of transfection efficiency using fluorescence microscopy.

Krzywinska E., Ferretti L, & Krzywinski J. (2022). Establishment and a comparative transcriptomic analysis of a male-specific cell line from the African malaria mosquito Anopheles gambiae. Scientific Reports, 12, 6885.

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CRISPR-Cas9 Plasmid Transfection in 293T Cells

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Plasmids were introduced into ~ 80% confluent 293 T cells in 24-well (2 cm²) plates using Lipofectamine™ 3000 (Life Technologies, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, 0.5 μg of gRNA-expressing pSpCas9(BB)-2A-GFP plasmid construct was diluted in 25 μL Opti-MEM reduced serum media (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1 μL of P3000 Reagent (Life Technologies). Then, 1.5 μl Lipofectamine 3000 reagent was diluted in 25 μL Opti-MEM and thereafter mixed with the diluted DNA/P3000 Reagent. The mixtures were incubated at room temperature for 5 min and thereafter added dropwise to each well of cells.

Kamali E., Rahbarizadeh F., Hojati Z, & Frödin M. (2021). CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells. BMC Biotechnology, 21, 9.

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Breast Cancer Cell Line Maintenance and Transfection

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This study used breast cancer cell lines obtained from ATCC, including ERα+ (MCF-7 and T47D) and ERα− (MDA-MB-231). The MCF-7 cells were maintained in Dulbecco’s Modified Eagle Medium (cat No. 11995, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (cat No. 16000, Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin (cat No. 15140, Invitrogen). The T47D and MDA-MB-231 cells were grown in Roswell Park Memorial Institute 1640 (Gibco, El Paso, TX) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were cultured at 37°C in the presence of 5% CO₂. The cells were starved for 24 hours and treated with ICI (1 to 10 μM, fulvestrant, Sigma-Aldrich, St. Louis, MO), IFN-γ (100 units/mL, R&D Systems, Minneapolis, MN), or β-estradiol (1 nM, Sigma-Aldrich) in 2 mL of medium for an appropriate time. The cells were then used in the protein expression assays.
For the ESR1 plasmid transfection, ERα-cells were plated and cultured in a 6-well plate at 90% confluency and transfected with 2.5 μg of hESR-GFP (cat No. #28230, Addgene, Cambridge, MA) using 3.75 μL of Lipofectamine 3000 reagent (Life Technologies) and 5 μL of P3000 reagent (Life Technologies) per well according to the manufacturer’s protocol.

Song I.H., Kim Y.A., Heo S.H., Bang W.S., Park H.S., Choi Y.H., Lee H., Seo J.H., Cho Y., Jung S.W., Kim H.J., Ahn S.H., Lee H.J, & Gong G. (2021). The Association of Estrogen Receptor Activity, Interferon Signaling, and MHC Class I Expression in Breast Cancer. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 54(4), 1111-1120.

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