To generate HNSCC cell lines stably expressing blue fluorescent protein (BFP), tandem-dimer Tomato (tdTomato), or enhanced green fluorescent protein (eGFP), HEK293 cells were used for lentivirus production. The cells were transfected, 24 h after plating, with calcium phosphate containing 2 μg psPAX2, 1 μg pMD2.G, and 2.5 μg pWPXL plasmid (Addgene, Watertown, Massachusetts, USA). The original green fluorescent protein (GFP) insert was replaced with either mTagBFP (blue fluorescent protein) or tdTomato. The viruses were collected at 24, 48, and 96 h after transfection, pooled, and passed through a 45 μm filter (Sarstedt). Between 100,000 and 200,000 HNSCC cells per well were plated in a 6-well plate one day before transduction individually defined per cell line. To obtain pure BFP and tdTomato-expressing cell populations, cells with the highest 5% intensity were isolated with fluorescence-activated cell sorting (FACS, BD FACSAria™ III; BD Biosciences, Franklin Lakes, NJ, USA). The stable expression of the fluorescent proteins was examined via flow cytometry and revealed a purity of 98.6% for Cal33-BFP, 97.7% for Cal33-tdTomato, 51.4% for FaDu-BFP, and 96.8% for FaDu-tdTomato cells.
Pwpxl plasmid
Manufactured by Addgene
Sourced in United States
The PWPXL plasmid is a DNA construct that serves as a vector for gene expression. It is commonly used in molecular biology research to introduce and express genetic material in various cell types.
Automatically generated - may contain errors
4 protocols using pwpxl plasmid
1
Generating Fluorescent HNSCC Cell Lines
2
Cloning and Lentiviral Transduction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-length human CD24A and CD24B open reading frame cDNA sequences were separately amplified and cloned into a pWPXL plasmid (Addgene, Cambridge, MA, USA) using BamHI and EcoRI. The full-length human EGR1 open reading frame cDNA sequence was amplified and cloned into pWPXL using MluI and EcoRI. The CD24A promoter, which spans a 2,100 bp-region (−1,900 bp to +200 bp based on the first ATG), and mutant were separately cloned into PGL3-Enhancer (Promega Corporation, Fitchburg, WI, USA). The primer sequences are listed in Table S1 .
For lentivirus production and cell transfection, after co-transfection of HEK 293 T cells with the pWPXL-CD24A vector, pWPXL-CD24B vector or pWPXL-EGR1 vector with psPAX2 and pMD2.G (Addgene) using Lipofectamine 2000 (Thermo Fisher Scientific) for 48 hours, viruses were harvested and used to infected target cells in the presence of 6 µg/mL polybrene (Sigma-Aldrich Co.).
For lentivirus production and cell transfection, after co-transfection of HEK 293 T cells with the pWPXL-CD24A vector, pWPXL-CD24B vector or pWPXL-EGR1 vector with psPAX2 and pMD2.G (Addgene) using Lipofectamine 2000 (Thermo Fisher Scientific) for 48 hours, viruses were harvested and used to infected target cells in the presence of 6 µg/mL polybrene (Sigma-Aldrich Co.).
3
Mitochondrial Membrane Potential and H2O2 Sensing
4
Lentiviral Transduction of Cox4i1/2
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!