DCA was purchased from Santa Cruz. 17-AAG was from Selleck. MG132 was purchased from Calbiochem. Cycloheximide, wortmannin, bafilomycin A, chloroquine and 3MA were purchased from Sigma-Aldrich. Wt and mutant p53 constructs were a gift from Dr. Shannon C Kenney. Transfection of wt p53 and R248Q was carried out using Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM (Invitrogen), according to the manufacturer’s instructions. p53 siRNA was a gift from Dr Xirodimas and it was ON-TARGETplus SMARTpools (mixture of 4siRNA) from Dharmacon. Two siRNA duplexes were used to knockdown Hsc70 as previously described50 (link). siRNAs were synthesized by Eurofins MWG Operon. Cells (2 × 106 in 100 μl) were transfected with 100 nM Hsc70 siRNA, p53 siRNA or control siRNA by electroporation using Amaxa SF Cell line 4D-Nucleofector kit (Lonza Bioscience). Cells were harvested 24 to 72 h post-transfection.
Amaxa sf cell line 4d nucleofector kit
Manufactured by Lonza
The Amaxa SF Cell line 4D-Nucleofector kit is a laboratory equipment product designed for the efficient and gentle transfection of a wide range of cell types, including suspension and adherent cell lines. The kit utilizes Nucleofection technology to deliver nucleic acids, such as plasmids or siRNA, into the cell nucleus, enabling gene expression studies and gene knockdown experiments.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine, by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
On targetplus smartpool, by Horizon Discovery (1 mentions)
Bafilomycin a, by Merck Group (1 mentions)
17 aag, by Selleck Chemicals (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Amaxa p3 primary cell 4d nucleofector kit, by Lonza (1 mentions)
2 protocols using amaxa sf cell line 4d nucleofector kit
1
Knockdown of Hsc70 and p53 in Cells
2
Introducing DSBs in Mammalian Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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To introduce DSBs in mammalian cells using an RNP complex, 2 × 105 cells were transfected with Cas9 protein (4.5–45 μg) premixed with in vitro transcribed sgRNA (6–60 μg). Cas9 protein in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol) was mixed with sgRNA dissolved in nuclease-free water and incubated for 10 min at room temperature. No more than 4 μL of the RNP mixture was added to 20 μL of the Nucleofection solution. For plasmid-mediated expression of RGENs, 2 × 105 cells were co-transfected with 1 μg of Cas9-encoding plasmid and 1 μg of sgRNA-expressing plasmid in K562 and BJ fibroblasts or 2.4 μg of Cas9-encoding plasmid and 1.6 μg of sgRNA-expressing plasmid in H9 hES cells. K562 cells were transfected with the Amaxa SF Cell Line 4D-Nucleofector Kit using Program FF-120 (Lonza), and H9 and BJ cells were transfected with the Amaxa P3 Primary Cell 4D-Nucleofector Kit using Program CB-150 and DT-130, respectively, according to the manufacturer’s protocol. Cells were analyzed 2 d after transfection, unless indicated otherwise.
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