Wild type HEK293 cells were kept in complete DMEM medium with Glutamax I (Invitrogen/LifeTechonologies, Darmstadt, Germany), 10% (v/v) fetal bovine serum (Biochrom, Berlin, Germany) and 100 U/l pencillin, 100 μg/l streptomycin (Invitrogen/ LifeTechonologies, Darmstadt, Germany) at 37°C and 5% CO2. For HEK293 cells stably expressing TRPM2 (clone #24) G418 sulfate (Biochrom, Berlin, Germany) was added to complete medium (final conc. 400 μg/ml).
G418 sulfate
Manufactured by Harvard Bioscience
Sourced in Germany
G418 sulfate is a broad-spectrum antibiotic commonly used as a selectable marker in cell culture experiments. It functions by inhibiting protein synthesis in eukaryotic cells, allowing for the selection of cells that have been successfully transfected or transduced with a gene of interest.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Venor gem mycoplasma detection kit, by Minerva Biolabs (1 mentions)
Htb 22, by American Type Culture Collection (1 mentions)
Crl 1619, by American Type Culture Collection (1 mentions)
P tdtomato, by Takara Bio (1 mentions)
Lenti x bicistronic expression system, by Takara Bio (1 mentions)
4 protocols using g418 sulfate
1
HEK293 Cell Culture Conditions
2
Generating Stable TRPM2-Expressing HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Generation
of the clonal HEK293 cell line with stable expression of human TRPM2
has been described previously.16 (link) In brief,
HEK293 were transfected with an expression vector encoding the full-length
of human TRPM2 and EGFP (pIRES2-EGFP-TRPM2). Cells that successfully
integrated the expression vector were then enriched by selection with
400 μg/mL G418 sulfate (Biochrom). Clonal cell lines were established
from these cells by limiting dilution. Expression of TRPM2 was confirmed
by Ca2+ measurement and whole cell patch clamp.
of the clonal HEK293 cell line with stable expression of human TRPM2
has been described previously.16 (link) In brief,
HEK293 were transfected with an expression vector encoding the full-length
of human TRPM2 and EGFP (pIRES2-EGFP-TRPM2). Cells that successfully
integrated the expression vector were then enriched by selection with
400 μg/mL G418 sulfate (Biochrom). Clonal cell lines were established
from these cells by limiting dilution. Expression of TRPM2 was confirmed
by Ca2+ measurement and whole cell patch clamp.
3
Metastatic Melanoma and Breast Cancer Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human metastatic melanoma cell line A375 (LGC Standards, ATCC® CRL-1619™, Wesel, Germany), transgenic A375 cells, and human breast adenocarcinoma cell line MCF-7 (LGC Standards, ATCC® HTB 22™) were cultured in Dulbecco´s Modified Eagle Medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin (PenStrep) (all reagents from Biochrom, Berlin, Germany) at 37 °C in a humidified atmosphere with 5% CO2. Transgenic cells were further cultured with 1.2 mg/mL G418 sulfate (Biochrom). All cells were routinely tested for mycoplasma contamination using Venor®GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). For proliferation assay, 5 × 104 A375-pIRES and A375-EphB4 cells were seeded into 6 well plates. After 1, 2, 3, 4, and 7 days cells were trypsinized and the number of viable cells was determined using a CASY®Model TT.
4
Generation of tdTomato and EpCAM-Expressing LL/2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A PCR product containing the sequence of tdTomato (vector ptdTomato; #63-2531, TaKaRa Clontech) was cloned into the lentiviral expression vector pLVX-IRES-neo (LentiX-Bicistronic Expression System; #63-2181, TaKaRa Clontech) to generate a pLVX-tdTomato-IRES-Neo construct. Notably, a resistance-sequence for G418-sulfate is contained in the lentiviral expression vector. The resulting nucleotide pLVX-tdTomato-IRES-Neo was verified by Sanger sequencing and restriction enzyme digestion.12 (link) LL/2 was transfected with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000; Thermo Fisher Scientific). tdtLL/2 was enriched by cultivation in selection medium containing G418-sulfate (#A2912; Biochrom) and by repetitive FACS sorting. As previously described in detail,13 (link) tdtLL/2 cells were stably transduced with a pMXs vector containing the full-length murine EpCAM (UNIPROT entry: #Q99JW5) cDNA to generate the EpCAM-overexpressing cell line EpCAM/tdtLL/2.
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!