Puromycin

Manufactured by Takara Bio

Sourced in United States, Japan

Puromycin is a laboratory reagent used in cell culture applications. It is an antibiotic that inhibits protein synthesis by interfering with translation, making it a useful tool for selection and maintenance of genetically modified cell lines.

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145 protocols using puromycin

Cell Line Maintenance and Tracking

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A549, HT1080 and HEK293T cells were obtained from our own stocks. MDA-MB-231 cells were gifted by Erle Robertson. These four cell lines were maintained in complete DMEM media containing 10% FBS and 1% penicillin/streptomycin. GFP (1–10) engineered versions of A549, HT1080, and HEK293T, which we previously described,^{23 (link)} and MDA-MB-231 GFP (1–10), which was made for this study, were maintained in the same media supplemented with 2 μg/mL puromycin (Takara Bio).
CT26 cells were obtained from Celeste Simon and maintained in RPMI containing 10% FBS and 1% P/S. CT26 GFP (1–10) cells were maintained in the same media supplemented with 8 μg/mL puromycin. HCT116 cells were gifted by Michael Farwell and were maintained in McCoy 5A media with 10% FBS and 1% penicillin/streptomycin. HCT116 GFP (1–10) cells, which we previously described,^{22 (link)} were maintained in the same media supplemented with 2 μg/mL puromycin. All cells were maintained in a 5% CO2, 37°C humidified incubator.

Kovbasnjuk O., Leader J.P., Weinstein A.M, & Spring K.R. (1998). Water does not flow across the tight junctions of MDCK cell epithelium. Proceedings of the National Academy of Sciences of the United States of America, 95(11), 6526-6530.

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Engineered UCMSCs Enhance Splenocyte Proliferation

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hUCMSCs were plated in 100 mm plates at 6 × 10⁶ cells per plate. After one day, the cells were 70% confluent. The cells were incubated sequentially with the 48 hour and 72 hour lentivirus-IL-21 and lentivirus supernatants respectively for 12 h. Following the last transduction, the cells were washed and incubated with fresh growth medium to allow puromycin-resistance expression. Two days later, puromycin selection was performed by incubating the cells in growth media supplemented with 1 μg/mL puromycin (Clontech Laboratories Inc.) for five days. After selection, hUCMSCs containing lentivirus-IL-21 (named hUCMSCs-LV-IL-21) and hUCMSCs lentivirus without IL-21 (named hUCMSCs-LV-Vec) were selected respectively. Then IL-21 expression was analyzed by western blotting. To identify the bioactivity of UCMSCs-LV-IL-21, the splenocytes from the normal nude mice were incubated with the supernatant from the cultured hUCMSCs-LV-IL-21 for 72 h, then the splenocyte proliferative activity was detected by FCM as described in a previous report
[27 (link)].

Zhang Y., Wang J., Ren M., Li M., Chen D., Chen J., Shi F., Wang X, & Dou J. (2014). Gene therapy of ovarian cancer using IL-21-secreting human umbilical cord mesenchymal stem cells in nude mice. Journal of Ovarian Research, 7, 8.

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Lentiviral Knockdown of Idh2 Gene

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Idh2 shRNA and nontarget shRNA MISSION® lentiviral transduction particles were purchased from Sigma-Aldrich. LLC cells were transduced with a final concentration of 8 μg/mL hexadimethrine bromide, according to the manufacturer's protocol. Transduced cells were selected as single colonies in a medium containing 5 μg/mL puromycin (Clontech, Mountain View, CA) and maintained in a medium containing 1 μg/mL puromycin.

Park J.H., Ku H.J., Lee J.H, & Park J.W. (2017). Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration. Oxidative Medicine and Cellular Longevity, 2017, 1595103.

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High-throughput mitophagy screening

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C2C12 cells were first infected with lentiviruses prepared from pLenti hParkin and pFUGW spCas9 (Addgene 52962) with 8 μg/mL polybrene. 24 hrs after infection, cells were treated with 2mg/ml Hygromycin (Mediatech) and 10 μg/mL Blastcidin (Invitrogen) for 4days and maintained 1 weak without drugs. Several single cell clones were developed and tested the ability to induce mitophagy. The well-induced clone was expanded and additionally infected with lentivirus prepared from pLenti Cox8-GFP, pLenti GFP-Omp25, or pLenti Cox4-mKeima, and then GFP or mKeima positive cells were sorted with narrow signal range. These cells were subsequently infected with the genome-wide GeCKO lentiviral library (Addgene 1000000053) at a MOI of ~0.5 (to yield ~100 cells per gRNA) with 8μg/mL polybrene in 6-well plates, with 0.6×10⁶ cells infected per well. Infected cells were treated with 2.5 μg/mL puromycin (Clontech) for 4 days, and the cells were expanded for an additional 6 days without puromycin. All virus infection was performed by centrifugation at 1000 g for 90 min, then the medium was removed, and cells were incubated in fresh medium for 24 hrs.

Hoshino A., Wang W., Wada S., McDermott-Roe C., Evans C.S., Gosis B., Morley M.P., Rathi K.S., Li J., Li K., Yang S., McMannus M.J., Bowman C., Potluri P., Levin M., Damrauer S., Wallace D.C., Holzbaur E.L, & Arany Z. (2019). The ATP/ADP translocase drives mitophagy independent of nucleotide exchange. Nature, 575(7782), 375-379.

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Lentiviral Transduction of JB6 Cells

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IDH2 short-hairpin RNA (shRNA) and non-target shRNA MISSION® lentiviral transduction particles were purchased from Sigma. JB6 cells were transduced with hexadimethrine bromide at a final concentration of 8 μg/mL, as per the manufacturer's protocol. Transduced cells were selected as single colonies in medium containing 5 μg/mL puromycin (Clontech, Mountain View, CA) and maintained in medium containing 1 μg/mL puromycin.

Park J.H., Ku H.J., Lee J.H, & Park J.W. (2018). IDH2 deficiency accelerates skin pigmentation in mice via enhancing melanogenesis. Redox Biology, 17, 16-24.

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High-throughput mitophagy screening

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Generating CHOP reporter cell line

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To generate a CHOP reporter stable cell line, PC 3 cells were co-transfected with 8 μg of a promoter construct (CHOP::GFP) [50 (link)] obtained from Addgene (Addgene plasmid 21898) and 800 ng of puromycin linearized selection marker (Clontech) using Lipofectamin (Invitrogen) per manufacturer’s instructions. Transfected PC3 cells were cultured in RPMI (Lonza) with 1 μg/ml puromycin (Clontech) for 3 weeks and colonies were picked using cloning cylinders. GFP expression was monitored in the IncuCyte™ imaging system (Essen Bioscience) at an excitation wavelength of 450–490 nm and an emission of 500–530 nm, and analyzed by Image J software (National Institutes of Health).

Park H.K., Lee J.E., Lim J, & Kang B.H. (2014). Mitochondrial Hsp90s suppress calcium-mediated stress signals propagating from mitochondria to the ER in cancer cells. Molecular Cancer, 13, 148.

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Generating Cav-2 Knockdown Hirc-B Cells

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Cav-2 shRNA stable Hirc-B cells were generated as described (33 (link)). Hirc-B cells were transfected with Cav-2 shRNA expressing plasmid (MISSION^® shRNA Bacterial Glycerol Stock, Sigma) for 48 h and subjected to incubation with 1 μg/ml puromycin (Clontech) to select for puromycin-resistant clones for a week. Independent colonies were then picked using cloning cylinder (Sigma), subcultured, and tested for Cav-2 expression by reverse transcription (RT)-PCR and immunoblot analysis. Stable cell lines that express a Cav-2 shRNA were then selected.

Jeong K., Kwon H., Lee J., Jang D, & Pak Y. (2015). Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane. Nucleic Acids Research, 43(6), 3114-3127.

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Genome editing protocol for XTP1 and CDK6

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The target gene was used as a template and evaluated using design software (http://tools.genome-engineering.org) to find the interference target sequence. The target sequence of XTP1 was 5'-GCTGCTAGATTGGTAACGTTT-3', and the target sequence of CDK6 was 5'-GCCCAACCAATTGAGAAGTTT-3'. The negative control sequence was 5'-TCTCCGAACGTGTCACGT-3'. Next, lentivirus construction and cell transfection were conducted according to a published protocol (16 (link)). The transfected cells with puromycin resistance was screened by culture medium with puromycin (Clontech, USA, 631305).

Li K., Ma R., Meng L., Wang Q., Cao J., Yuan D., Sun T., Kang L., Hao N., Wang H, & Zhu K. (2023). XTP1 facilitates the growth and development of gastric cancer by activating CDK6. Annals of Translational Medicine, 11(2), 97.

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Stable ADAR1 Knockdown in RPTECs

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RPTECs were infected with lentiviral particles expressing small hairpin RNA (shRNA) against the human ADAR1 gene (sc-37657-V; Santa Cruz Biotechnology), which contained three target-specific constructs encoding 19 to 25-nucleotide shRNA designed to repress the expression of ADAR1. To select clones stably expressing shRNA, cells were maintained in a medium containing 5 μg/ml of puromycin (FUJIFILM Wako Pure Chemical Corporation). Downregulation of ADAR1 was confirmed by Western blotting. To construct mock-transduced RPTECs as control cells, naive RPTECs were infected with lentivirus particles derived from pLVSIN-CMV Pur vector (Takara Bio Inc) and were cultured in puromycin-containing medium as described above to select the stably expressing cells.

Omata Y., Okawa M., Haraguchi M., Tsuruta A., Matsunaga N., Koyanagi S, & Ohdo S. (2022). RNA editing enzyme ADAR1 controls miR-381-3p-mediated expression of multidrug resistance protein MRP4 via regulation of circRNA in human renal cells. The Journal of Biological Chemistry, 298(8), 102184.

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