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26 protocols using Puromycin

1

Transfection and Encystation of Giardia and Spironucleus

Plasmid DNA for transfection of S. salmonicida or G. intestinalis was prepared as described in reference [20] (link). Culture and transfection of S. salmonicida and G. intestinalis WB/C6 were according to reference [20] (link) and reference [75] (link), respectively. Transfectants of either organism were selected and maintained using 50 µg/ml puromycin (A.G Scientific). The G. intestinalis transfectants were induced to encyst by replacing the normal growth media of 70–80% confluent cultures with encystation media with pH 7.8 and 1.25 mg/ml of bovine bile. In vitro generated cysts were harvested 48 h post induction by centrifugation at 500× g and kept in water at 4°C for 24 h.
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MCF10A cells (ATCC) were maintained in DMEM/F12 medium (Thermo Fisher Scientific 11-330-057) with 5% horse serum (Thermo Fisher Scientific 26050088) that was supplemented with 10 μg/ml insulin (ThermoFisher Scientific 12585014), 10 ng/ml EGF (PeproTech AF-100-15), 0.5 μg/ml hydrocortisone (Sigma-Aldrich H4001), and 100 ng/ml cholera toxin (Sigma-Aldrich C8052). MDA-MB-231 GFP-LUC cells 23, 24 (link) were maintained in DMEM (Corning 10-017-CV) with 10% FBS (Gemini Bio-Products 100-106). MCF10A, MCF10A PTEN -/-38 , MCF10A KRas(G12V) 23 , and MCF10A KRas(G12V)/PTEN -/-23 cells were infected with a LifeAct-GFP expressing lentivirus; LifeAct cells were used in all comparisons between these four cell lines reported in this work. All LifeAct cells were cultured in MCF10A media supplemented with 0.5 μg/ml puromycin (AG Scientific P-1033-sol). All cells were maintained in a humidified atmosphere at 37 °C and 5% CO2. All cells used in this study tested mycoplasma negative using the Lonza MycoAlert (VWR 75870-454) testing system.
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The detailed cell culture method was described previously 7. Huh‐7.5 cells (Apath, LLC, Brooklyn, NY, USA) and HepG2 cells (ATCC, Manassas, VA, USA) were maintained in DMEM supplemented with 10% fetal bovine serum, 4.5 g/l glucose, L‐glutamine and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). shRNA‐harbouring cells were cultured in complete medium supplemented with 1 μg puromycin (Sigma‐Aldrich, St. Louis, MO, USA), and shRNA‐resistant DGAT1‐transfected, DGAT1 knock‐down cells were maintained in complete medium supplemented with 1 μg puromycin and 1 mg/ml G418 (A.G. Scientific, San Diego, CA, USA).
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To generate mCherry- or mPlum-expressing MCF-7 breast tumor cells, retroviral infections were performed as previously described [40 (link)]. Briefly, the mCherry-pQCXI or mPlum-pQCXI plasmids (each with a puromycin selection marker) were transfected by calcium phosphate to HEK293T cells (a generous gift from Dr. Bacharach, Tel Aviv University) together with plasmids encoding gag-pol and VSV-G proteins. Supernatants were collected after two days, filtered through a 0.45-μm mesh and incubated with MCF-7 cells in the presence of 8 μg/ml polybrene for 5 hrs. The infection process was repeated on the following day, in order to increase infection yields. Three days following the second infection, infected cells were selected in 8 μg/ml puromycin (#P-1033; A.G. Scientific, San Diego, CA) for seven days.
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We purchased pBABE-hLin28B plasmid (Addgene, 26358) that expresses FLAG-tagged human LIN28B protein24 (link). We generated AluJb-LIN28B CDS from H1299 mRNA and cloned AluJb-LIN28B CDS in lieu of FLAG-hLIN28B sequence into the pBABE vector. We co-transfected pBABE plasmids with pMD2.G and pUMVC following polyethylenimine (PEI) transfection protocol into HEK293T cells. AluJb KO clones were transduced with viral supernatant supplemented with polybrene (5 μg/ml) for two days. Successfully infected cells were selected by 2 μg/ml puromycin (A.G. Scientific, P-1033-sol) treatment for 5 days before subsequent analysis.
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Viral particles were produced in HEK293LV cells using the 2nd Generation packaging and envelope plasmids psPAX2 and pMDG.2 (Addgene). Virus production and transfection followed the manufacturer's instructions (http://www.addgene.org/tools/protocols/plko/#E). MDA-MB-231 cells were transfected using Viafect (Promega) at a ratio of 4:1 (Viafect:DNA) with pLenti-EF1α-CymR-Neo vector (ABM, Richmond, Canada) containing the cumate repressor and neomycin resistance genes. Selection was carried out using G418 (Sigma) at 500 μg/ml. Once cloned, cells were further transfected with the pLenti GFP-profilin construct and stably transfected cells were selected with 1 μg/ml puromycin (AG Scientific, San Diego).
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7

Lentiviral Transduction and Stable Cell Line Generation

HeLa, 293T, HepG2 and SW480 cells were cultured in DMEM containing 10% FBS. Lentiviral supernatants were produced using Lenti-X Packaging Single Shots (VSV-G) (Clontech #631275) mixed with pLKO.1 puro-based lentiviral constructs transfected to Lenti-X 293T cells. 24 hours after transfection, supernatants were collected and concentrated using Lenti-X Concentrator (Clontech #631231). Stable shRNA-expressing cell lines were established by infecting cells with shRNAcontaining lentiviral supernatants together with 4 μg/ml protamine sulfate for 24 hours. After 24-48 hours of culture with fresh medium, cells were selected with medium containing 1~2 μg/ml puromycin (AG Scientific #P-1033) until non-transduced cells became completely dead, which usually takes 2-3 days. Plasmid DNAs were transfected using Fugene HD (Promega #E2312) according to the manufacturer's protocol.
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We purchased pBABE-hLin28B plasmid (Addgene, 26358) that expresses FLAG-tagged human LIN28B protein24 (link). We generated AluJb-LIN28B CDS from H1299 mRNA and cloned AluJb-LIN28B CDS in lieu of FLAG-hLIN28B sequence into the pBABE vector. We co-transfected pBABE plasmids with pMD2.G and pUMVC following polyethylenimine (PEI) transfection protocol into HEK293T cells. AluJb KO clones were transduced with viral supernatant supplemented with polybrene (5 μg/ml) for two days. Successfully infected cells were selected by 2 μg/ml puromycin (A.G. Scientific, P-1033-sol) treatment for 5 days before subsequent analysis.
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Vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentivirus was made by co-transfecting HEK-293T cells with the pLenti CMV/TO constructs along with PAX2 and VSV-G plasmids. Viral supernatants were collected and equal amounts of the supernatants were used to infect ARPE-19 TR (“Tet-On”) cells (described previously33 (link)). Stable, heterogeneous cell populations were generated by selection with puromycin (1 μg/mL; A.G. Scientific, San Diego, CA, USA) for >2 weeks. Cell cultures were routinely screened for mycoplasma (MycoAlert Plus, Lonza, Walkersville, MD, USA). Stable NFκB ARPE-19 reporter cells were generated by cotransfecting cells with a plasmid encoding for 5xNFκB responsive elements driving the expression of a secreted luciferase, Gaussia luciferase (GLuc, plasmid called “5NF-GLuc” here onward, described previously34 (link)) and a constitutively expressed green fluorescent protein (GFP) along with a pLKO puro shSCRAM vector (Sigma-Aldrich Corp., St. Louis, MO, USA) followed by puromycin selection and flow cytometry–assisted cell sorting based on GFP signal. Human IL-1α and human TNFα were purchased from PeproTech (Rocky Hill, NJ, USA), LPS was purchased from Sigma-Aldrich Corp., 4-HNE was purchased from Enzo (Farmingdale, NY, USA), and A2E was purchased from Gene and Cell Technologies (Vallejo, CA, USA).
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The SCOTIN‐KO cell line was generated using SCOTIN‐KO and HDR plasmids (Santa Cruz Biotechnology, sc‐408226, sc‐408226‐HDR) following the manufacturer's protocol. The two plasmids were transfected into HeLa cells and cultured in medium containing puromycin (1 μg/ml; AG Scientific, P‐1033‐SOL). Each colony was analyzed for the depletion of endogenous expression by Western blotting.
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