Lentiviral vectors were generated at the UCLA Integrated Molecular Technologies Core (Figure 1 A). Regarding c-MYC and the large T antigen of the Simian virus 40, plasmids for the lentiviruses were obtained from Addgene (pcDNA3-c-MYC, #16011 and pBSSVD2005, #21826, respectively, Watertown, MA, USA). Each gene was packaged in a lentivirus backbone with a puromycin resistance gene, pac. Primary CSSCs, from the same tissue donor, at passage 2 (50–60% confluence, 6-well plate well) were transduced by the appropriate viral particles with a final particle amount of 0.54–0.58 µg for 24 h. Cells were washed with PBS and incubated with fresh medium. Three days later, puromycin selection (1 µg/ml) was started for 15 days, with medium renewal every 3 to 4 days.
Pbssvd2005
Manufactured by Addgene
Sourced in United States
The PBSSVD2005 is a lab equipment product. It is a vacuum desiccator designed for drying and storage of samples. The core function of this product is to provide a controlled environment for preserving samples by removing moisture.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep antibiotic, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium high glucose, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Isotopically labeled lysine and arginine, by Cambridge Isotopes (1 mentions)
Silac dmem media, by Thermo Fisher Scientific (1 mentions)
6 protocols using pbssvd2005
1
Lentiviral Transduction of Primary CSSCs
2
Plasmid-mediated Gene Expression
3
Generation of Hat1-Deficient Mouse Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse embryonic fibroblasts (MEFs) derived from embryos obtained from crosses of Hat1+/− males and females. Pregnant heterozygous female mice were euthanized at 13.5dpc, and the embryos were cultured to generate Hat1+/+, Hat1+/−, and Hat1−/− fibroblasts. These MEFs were cultured in Dulbecco's modified Eagle's medium–high glucose (Sigma) supplemented with 10% fetal bovine serum (FBS) and 1X penicillin/streptomycin antibiotics. SV40 T immortalized MEFs (iMEFs) were derived from primary Hat1+/+ and Hat1−/− embryonic day 13.5 embryos. To establish iMEFs, early passage cells were transformed with SV‐40T antigen containing plasmid pBSSVD2005 (ADDGENE). Early passage cells were seeded at 25% confluency in 6‐well plates and transfected with 2 μg of expression vector using Fugene reagent (Roche). Cells were harvested and seeded into 100‐mm dishes after 48 hr of transfection. The cells were split at 1 in 10 dilutions until passage 5.
4
SV40-Immortalized MEF Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E14.5 embryos were dissected from uterus; head, limbs and internal organs were removed. Tissue was disaggregated using an 18-gauge syringe and brought to single cell suspension with trypsin incubation at 37°C. Cells were then plated onto 100 mm tissue culture plates, passaged upon confluency and maintained in Dulbecco's modified Eagle medium (DMEM-Sigma) with 10% fetal bovine serum (FBS-Gibco) and 1X Pen/Strep antibiotics (Sigma). SV40 T immortalized MEFs (iMEFs) were derived from primary Hat1+/+ and Hat1−/− embryonic day 13.5 embryos. To establish iMEFs, early passage cells were transformed with SV-40 T antigen containing plasmid pBSSVD2005 (ADDGENE, Cambridge, MA). Early passage cells were seeded at 25% confluency in six-well plates and transfected with 2 ug of expression vector using Fugene reagent (Roche). Cells were harvested and seeded into 100 mm dishes after 48 h of transfection. The cells were split at 1 in 10 dilutions until passage 5.
For the SILAC experiments, cells were grown in SILAC DMEM media (ThermoFisher Scientific) and supplemented with 10% dialyzed fetal bovine serum (ThermoFisher Scientific), antibiotics and isotopically labeled lysine and arginine (Cambridge Isotope laboratories). Hat1−/− cells were grown under these conditions for at least four passages to guarantee the full incorporation of the labeled amino acids.
For the SILAC experiments, cells were grown in SILAC DMEM media (ThermoFisher Scientific) and supplemented with 10% dialyzed fetal bovine serum (ThermoFisher Scientific), antibiotics and isotopically labeled lysine and arginine (Cambridge Isotope laboratories). Hat1−/− cells were grown under these conditions for at least four passages to guarantee the full incorporation of the labeled amino acids.
5
Generation of Immortalized Mouse Embryonic Fibroblasts
6
Immortalization of Primary Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Patient’s primary fibroblasts were obtained by cultivating skin biopsies in RPMI 1640 supplemented with 20% FBS, 1% penicillin/streptomycin, 1X gentamicin, and 1X amphotericin B. Primary fibroblasts were transformed by lipofection of pBSSVD2005 (Addgene #21826) coding the large-T antigen of Simian virus-40. After 3 to 4 cycles of cultures, fibroblasts change of their shape and growing rate and were considered immortalized. This was later confirmed by evaluating expression of SV40 by confocal microscopy. SV40-fibroblasts were then maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.
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!