A pcDNA6/TR vector (Thermo Fisher Scientific) was introduced into SH-SY5Y cells for high-level expression of the tetracycline repressor protein. pcDNA6/TR vector was linearized with FspI restriction enzyme (NEB), purified with PCR Clean-up (Macherey-Nagel) and used for the transfection of SH-SY5Y cells using Xfect transfection reagent (TaKaRa) according to manufacturer's instructions. For 48 h, cells were exposed to selection medium containing tetracycline-free FBS (Gibco) and blasticidine S at 5 µg mL -1 (Sigma-Aldrich). Single-cell cloning of resistant cells was performed with dilution plating, followed by cell sorting for the homogenous expression of repressor proteins. To test the efficiency of repression, cells were transfected with a plasmid containing eGFP under a Tet operator. As the resulting SH-SY5Y-TR-eGFP cells exhibited no residual eGFP expression upon the addition of 1 µg mL -1 of doxycycline (Sigma-Aldrich), the original SH-SY5Y-TR cells were selected for FlpIn SH-SY5Y-TR-FRT cell line development. To introduce the FRT recombination site into the cells, pFRT/ lacZeo was linearized with ApaI (NEB) restrictase, purified with PCR Clean-up and transfected into SH-SY5Y-TR cells (Xfect, TaKaRa). Forty-eight hours later, cells were subjected to a
Blasticidine s
Manufactured by Merck Group
Blasticidine S is a lab equipment product manufactured by Merck Group. It is a chemical compound used in laboratory research and experiments. The core function of Blasticidine S is to serve as a selective antibiotic agent for certain types of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Xfect transfection reagent, by Takara Bio (1 mentions)
Pcdna6 tr vector, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Xfect, by Takara Bio (1 mentions)
Fspi restriction enzyme, by New England Biolabs (1 mentions)
Pcr clean up, by Takara Bio (1 mentions)
Tetracycline free fbs, by Thermo Fisher Scientific (1 mentions)
Pcr clean up, by Macherey-Nagel (1 mentions)
Hela 229, by American Type Culture Collection (1 mentions)
Human melanocyte growth supplement, by Thermo Fisher Scientific (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
A2058, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
2 protocols using blasticidine s
1
Tetracycline-Inducible SH-SY5Y Cell Line
2
Culturing Diverse Cell Lines for Experiments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293T, A2058 and GAK cells were obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). SKMEL28, G361, HMV1 and CRL1579 cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan. HeLa229 and U2OS cells were obtained from American Type Culture Collection. Cells were cultured in Dulbecco's Eagle's medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). Human Epidermal Melanocyte cells (Invitrogen) were cultured in Medium 254 (Invirogen, Portland, OR, USA) supplemented with Human Melanocyte Growth supplement (Invitrogen). All cells were cultured at 37°C in a humidified incubator under 5% CO2. FLAG-PITX1 stable expression clones were maintained in DMEM supplemented with 10% FBS and G418 (HeLa229 and U2OS 500 μg/ml, 293T 600 μg/ml; Calbiochem, La Jolla, CA, USA). FLAG-ZCCHC10 and PITX1 stable co-expression A2058 clones were maintained in DMEM supplemented with 10% FBS and G418 (300 μg/ml, Calbiochem) and blasticidine S (3 μg/ml, sigma). All Cell lines detected as Mycoplasma-free by MycoAlert mycoplasma detection kit (Lonza, Walkersville, MD, USA) and no more than 20 passage from the validated stocks.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!