K562 puromycin kill curve 1 vector. The concentration for selection should be determined beforehand with a kill curve. Higher doses will kill all cells after 2-3 days, and low doses will have minimal toxicity after the 7 days. K562 This project is supported by TOKU-E which specializes in manufacturing ultra-pure antibiotics for a broad spectrum of biotechnology applications as well as for the pharmaceutical industry. Cell . In this protocol, we highlight the use of puromycin, the antibiotic used with the standard pLKO. This is a crucial step before using a selection antibiotic to kill untransfected cells and generate stable cell We determined purmomycin concentration for K562 cells using the standard kill curve protocol recommended by Thermo Fisher Scientific. Kill curve/Puromycin DiHCl Titration: Seed cells of the parental cell line in a 24-well plate at different densities (50,000 – 100,000 and 200,000 cells/ml) and incubate the cells for 24 hours at 37°C. , puromycin). A kill curve is a dose-response experiment where the Access the Kill Curve Protocol from BPS Bioscience for accurate measurement of cell viability and cytotoxicity. TOKU-E is a global leader in biotechnology innovation, offering great benefits and applications to the biopharmaceutical and diagnostic industries as well as for biotechnology research communities. 2. g. Since mammalian cells sensitivity to antibiotics varies from one cell type to another, it is essential to determine the minimum concentration of antibiotic required to kill non-transfected or non-transduced cells by separate kill curve experiments. 2. So for the Kill Curve for puromycin you can use the concentration range of 0 - 20ug/ml puromycin on normal K562 cells. The 'kill concentration' is the lowest concentration of puromycin which results in cell dealth of approximately 100% after 72 hours + 1μg/ml to account for difference in scale up. It is a potent translational inhibitor in both prokaryotic and eukaryotic cells. Figure 2. Production of K562 Clones Hyper-Expressing α-Globin For the production of K562 cell clones with integrated copies of a α-globin-expressing vector, we transfected K562 cells with the pcDNA3. Puromycin selection at low concentrations Puromycin has a fast mode of action, causing rapid cell death at low antibiotic concentrations. For example, the 'kill concentration in Fig. Selection: 48 hours post-transduction, begin selection with the appropriate antibiotic (e. Plate cells in 0. In mammalian cells, the recommended working concentration range for puromycin is 0. In order to generate a stable cell line expressing a transgene or shRNA of interest, it is important to determine the minimum concentration of antibiotic required to kill non-transfected (plasmid DNA) or non-transduced (e. This detailed protocol helps researchers assess the effectiveness of treatments and compounds in cellular models. This is accomplished by performing a kill curve to determine the optimal puromycin concentration needed to eliminate non-transduced cells. Dose response curve for antibiotic selection of mammalian cells (kill curve) Mammalian cell sensitivity to antibiotics varies from one cell type to another. Protocol 1. A kill curve is a dose-response experiment where the Dose response curve for antibiotic selection of mammalian cells (kill curve) Mammalian cell sensitivity to antibiotics varies from one cell type to another. Optimization of puromycin selection condition To generate a fully transduced population of cells it is important to determine the minimum amount of puromycin required to eliminate non-transduced cells. Perform a kill curve to determine the optimal working Optimization of puromycin selection condition To generate a fully transduced population of cells it is important to determine the minimum amount of puromycin required to eliminate non-transduced cells. , lentiviral Jul 15, 2025 · In order to avoid the enrichment of double integrants and loss of single-integrants, we recommend avoiding over-selection: calculate an antibiotic kill curve in your cell line beforehand and choose the lowest antibiotic concentration which gives 90%-95% killing. Jan 29, 2019 · This curve establishes a kill curve to determine the antibiotic concentration required to kill cells that did not integrate the plasmid DNA. Different cell types and cell culture conditions may require different concentrations of selection antibiotic. Feb 13, 2018 · If both conditions are not well balanced, untransformed parental cells can survive or transfected cells may be killed. The optimal dose is the lowest antibiotic concentration where all cells are dead after 7 days. Typically, 1-10 µg/mL are sufficient to kill most untransduced mammalian cell types. 5 – 10 μg/ml. Kill curve for a puromycin titrated cancer cell line. 2 is 3μg/ml. Resistance to puromycin is conferred by the puromycin N-acetyltransferase gene (pac) from Streptomyces. 1 However, this kill curve simply defines the ideal concentration of SA needed to kill the untransfected cells of a particular cell line in 7–10 days of selection. For K562 cells, 2 μg/ml puromycin in growth media is optimum for selection for stable shRNA K562 cells. This general protocol can also be used for determining the optimal concentration of G418 required for the custom shRNA vectors. 1/Hygro (+)/α-globin vector and then selected positive cells by limiting dilutions in the presence of Hygromycin B selection antibiotic. Puromycin antibiotic ensures effective positive selection of cells expressing the puromycin-N-acetyl-transferase (pac) gene. , lentiviral The antibiotic kill curve is a dose-response assay used to select stable gene cell lines. What is an antibiotic kill curve? Antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic to determine the minimum concentration of an antibiotic that can kill all the cells in a specific period of time. 5 mL complete medium per well in 24-well plate. bnnlae ymngqf vgrdt msqy qpgzwnb nhj xmjl mpuuf nofljc weahwp enqz yqfmey gtzzoym uvmbc hafhr