Packaged lentivirus constructs (pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO) encoding CDK5RAP2 shRNA (TGGAAGATCTCCTAACTAA) or control shRNA were generated by Hanbio (Shanghai, China). Cells were seeded into 24-well plates and allowed to grow to 30–50% confluence, and then culture media mixed with shRNA lentiviral particles were added to the wells and the plates were incubated for 24 h. To select for infected cells, the cells were cultured in medium containing 10 μg/mL puromycin dihydrochloride (MedChemExpress) for ~72 h, after which the selected stable cells were cultured in medium supplemented with 0.5 μg/mL puromycin dihydrochloride. CDK5RAP2 knockdown efficiency was determined through Western blotting.
Puromycin dihydrochloride
Manufactured by MedChemExpress
Sourced in United States, China
Puromycin dihydrochloride is a laboratory reagent used as a selection marker in cell culture experiments. It functions as an antibiotic that inhibits protein synthesis, allowing for the selection of cells that have been successfully transfected or transduced with a puromycin resistance gene.
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Lab products found in correlation
4 protocols using puromycin dihydrochloride
1
Lentivirus-Mediated CDK5RAP2 Knockdown
2
Generating Stable CDCA2 Knockdown Cell Lines
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Huh7 and SK-Hep1 cells were both infected with lentivirus carrying the GV493 vector with CDCA2-specific shRNA (shCDCA2, target sequence: gcAAACCTTTCAGAGGAGAAA) or a nontargeting control sequence (shNC, sequence: TTCTCCGAACGTGTCACGT) at MOIs of 5 and 30, respectively, and uninfected cells were used as the control. After three days of growth, the cells were observed with a fluorescence microscope to confirm the expression of EGFP in >90% of cells. Puromycin dihydrochloride (MedChemExpress, Monmouth Junction, NJ, USA) was added to the complete growth media at a final concentration of 8 µg/mL for Huh7 cells and 6 µg/mL for SK-Hep1 cells, and this medium was refreshed every 3 days until all the uninfected cells had died and no more death occurred in the infected cells. The cells were then digested and seeded in 96-well plates at a concentration of 1 cell/well and continued to grow until stable strains were obtained.
3
Generation of Caspase-8 Lentiviral Cell Lines
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For lentiviral production (caspase-8WT and caspase-8K14R), the full-length complementary DNA of isoform 7 of caspase-8 containing rs3769823[A] or rs3769823 [G] was cloned into the pSLenti-SFH-EGFR-P2A-Puro-CMV-3xFLAG-WPRE vector. The lentivirus was purchased from OBIO Technology (Shanghai, China). For infection, cells were stably transfected with the lentivirus. Afterward, the cells were cultured in medium containing puromycin dihydrochloride (MedChemExpress). Finally, monoclonal stable transgenic cell lines were selected for in vivo assays.
4
Knockdown of TFRC Induces Ferroptosis in HUVECs
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HUVECs were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco's modified Eagle's medium supplemented with 10 % fetal bovine serum and 1 % (v/v) penicillin/streptomycin in a 37 °C incubator with a humidified atmosphere of 5 % CO2. The passage number of the cell lines was approximately F3∼F5.
A shRNA targeting TFRC was synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China), and the sequence that was used for interference was as follows: GCTGGTCAGTTCGTGATTAAA. The vector structure is shown inFig. S4C . HUVECs were transfected with TFRC shRNA or negative control shRNA using polybrene reagent. The cells were transduced with lentivirus (multiplicity of infection = 100). After three days, stable clones expressing the shRNA were selected via puromycin dihydrochloride (MedChemExpress, China). The medium was replaced with new puromycin-supplemented medium every 2–3 days until resistant colonies were selected. The resistant colonies were expanded, and GFP fluorescence and Western blotting analyses were performed to evaluate stable shRNA transduction and knockdown efficiency. Normal cells and transinfected cells were treated with DMSO (vehicle reagent), erastin (ferroptosis agonist, HY-15763; MedChemExpress), or deferoxamine mesylate (iron chelator, DFOM; HY-B0988; MedChemExpress) for 24 h.
A shRNA targeting TFRC was synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China), and the sequence that was used for interference was as follows: GCTGGTCAGTTCGTGATTAAA. The vector structure is shown in
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