Plasmids containing shRNAs sequences for SPAK and OSR1 form the Sigma Mission library (PLKO) were purified from bacterial glycerol stocks (Table S2 ). Lentiviral particles (LVPs) were produced using a standardized protocol in the laboratory. It requires the transfection of HEK293T cells (ATCC® CRL-3216) with the transfer plasmid of interest, the packaging plasmid (psPAX2 Addgene), and the envelope vector (pMD.2 Addgene) in the presence of Lipofectamine 3000 (Thermo Scientific). The LVPs were then concentrated using Lenti-XTM Concentrator (Clontech) and resuspended in PBS for quantitative characterization by ELISA using the Lenti-X p24 Rapid Titer Kit (Takara). GBM cell 965 was transduced and selected using puromycin (Invitrogen). The knockdown efficiency was corroborated by obtaining lysates and performing WB using antibodies specific for OSR1 (Cell Signaling-#3729), SPAK (Cell Signaling-#2281).
Pmd 2
Manufactured by Thermo Fisher Scientific
The PMD.2 is a compact and versatile lab equipment designed for precision measurement and data analysis. It provides accurate and reliable performance for a wide range of laboratory applications. The core function of the PMD.2 is to facilitate precise data collection and monitoring through its advanced sensing capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Pspax2, by Thermo Fisher Scientific (3 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Lenti x p24 rapid titer kit, by Takara Bio (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Lenti xtm concentrator, by Takara Bio (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
3 protocols using pmd 2
1
Lentiviral Knockdown of SPAK and OSR1 in GBM Cells
2
Lentiviral Knockdown of SPAK and OSR1 in GBM Cells
3
Lentivirus Production and Transduction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For lentivirus preparation, HEK293T cells were plated 1 day ahead to reach about 70% confluency for transfection, and VSV-G envelope expressing plasmid pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) were used for lentiviral packaging. Plasmids (1.8 μg) with the gene of interests were mixed with psPAX2 (1.35 μg) and pMD2.G (0.45 μg) for each well of six-well plates, and Lipofectamine® 3000 Reagent (Thermo Fisher Scientific, L3000) was used for transfection. Five to eight hours later, the medium was changed to fresh MEF medium. Supernatant containing the virus was harvested at 48 hours, passed through a 0.45-μM filter to remove the cell debris, and mixed with 1 volume of fresh medium for immediate use.
For infection, MEFs or NPCs were incubated with the lentiviral supernatant in the presence of 5 μg/ml polybrene (Millipore) for 8 hours or overnight. Medium was changed back to MEF or NPC medium after the infection for cells to recovery.
For infection, MEFs or NPCs were incubated with the lentiviral supernatant in the presence of 5 μg/ml polybrene (Millipore) for 8 hours or overnight. Medium was changed back to MEF or NPC medium after the infection for cells to recovery.
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!