The oxidation state of the mitochondrial GSH pool was assessed using a previously developed genetically encoded fluorescent and ratiometric biosensor targeted to the mitochondria35 (link) (Mito-Grx-roGFP2). The Mito-Grx1-roGFP2 construct (Addgene, 64977) was cloned into the pLenti-CMV-Puro vector (Addgene, 17448) and lentivirus generated to produce stable cell lines expressing the biosensor as described. For each treatment condition, 15,000 cells were seeded overnight onto triplicate wells of a black walled 96-well plate. Cells were then washed with PBS and treated with cystine replete or deficient RPMI for 16 h. Cells were then placed in a fluorescence-compatible plate reader (Promega, Madison, WI, USA) and fluorescence measured (GSH-bound Ex:475 nm/Em:500–550 nm, GSSG-bound Ex:405 nm/Em:500–550 nm).
Plenti cmv puro vector
Manufactured by Addgene
Sourced in United States
The PLenti-CMV-Puro vector is a lentiviral expression vector that allows for the stable integration and expression of a gene of interest in target cells. The vector contains a Cytomegalovirus (CMV) promoter to drive high-level expression of the gene of interest, as well as a puromycin resistance gene for selection of transduced cells.
Automatically generated - may contain errors
Lab products found in correlation
Repli g wta single cell kit, by Qiagen (1 mentions)
Puromycin, by Merck Group (1 mentions)
Puromycin, by Qiagen (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pcdh cmv mcs ef1 puro vector, by System Biosciences (1 mentions)
Pcdna3 flag traf6, by Addgene (1 mentions)
3 protocols using plenti cmv puro vector
1
Mitochondrial GSH Redox Biosensor
2
Spike-in Assay for Quantifying CTC
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For checking the specificity of the strategy, a modified Cal 27 cell line was used for spike-in experiments.
Cal 27 cells were transduced with the pLenti CMV puro Vector (#17448, Addgene, Cambridge, MA, USA) inserted with CRNN gene. This vector contains the puromycin resistance gene as a selection marker. Transduced cells were then selected with puromycin (5 µg/mL) (#P8833, Sigma) for 7–10 days. The detailed protocol is provided in theSupplementary Material
The modified Cal 27 cell line was spiked into whole blood in different concentrations of 10 cells/mL, 100 cells/mL, and 1,000 cells/mL. These spiked samples were processed for standardised CTC isolation protocol (PBMC isolation → CD45 depletion → sorting of cells using CTC isolation gating strategy). The sorted cells were subjected to whole transcriptome amplification using REPLI-g WTA Single Cell Kit (#150063, Qiagen) followed by qRT-PCR using SYBR green chemistry, and mRNA expression of the puromycin resistance gene was detected.
Cal 27 cells were transduced with the pLenti CMV puro Vector (#17448, Addgene, Cambridge, MA, USA) inserted with CRNN gene. This vector contains the puromycin resistance gene as a selection marker. Transduced cells were then selected with puromycin (5 µg/mL) (#P8833, Sigma) for 7–10 days. The detailed protocol is provided in the
The modified Cal 27 cell line was spiked into whole blood in different concentrations of 10 cells/mL, 100 cells/mL, and 1,000 cells/mL. These spiked samples were processed for standardised CTC isolation protocol (PBMC isolation → CD45 depletion → sorting of cells using CTC isolation gating strategy). The sorted cells were subjected to whole transcriptome amplification using REPLI-g WTA Single Cell Kit (#150063, Qiagen) followed by qRT-PCR using SYBR green chemistry, and mRNA expression of the puromycin resistance gene was detected.
3
Molecular Cloning of TRAF and FYN-TRAF3IP2 Constructs
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