Optimizing in vitro mammalian cell electrotransfection
Optimizing in vitro mammalian cell electrotransfection
Electrotransfection can be used for studies of gene function, promoter activity, and gene regulation; it can also be used to create transgenic or knockout embryonic stem cells and to deliver tumor antigens encoding R N A into dendritic cells for tumor vaccine research. Electrotransfection can also be used to deliver therapeutic genes to immune cells for tumor therapy or to stem cells for the treatment of different types of diseases Author: T. Friedman et al.
Operation method
Electrotransfection of mammalian cells Move Electrotransfection of mammalian cells material reagents Methods Preparation of cells to be electroporated 1 . 24-72 h before electroporation, isolate the cells and culture them in suitable complete medium containing serum. 2 - When the cultured cells reach mid or end logarithmic growth (or 60% to 90% coverage), collect the cells. For adherent cells, digest with IX Trypsin-EDTA solution (preheated to 37C) and then wash once with PBS or other wash buffer at room temperature. 3. Collect the cells by centrifugation at 250 g for 5 m i n at 20°C. Resuspend the cells with electroporation buffer to a cell density of 5X 105 ~ 5X 106 cells per 100 ul. 4 . Mix 2~IOug DNA and IOOmI cell suspension and transfer to an electroporation tube. Although there are times when it is recommended to depyrogenate the mixture of cells and D NA prior to electroporation, it is not necessary. This step should be performed at room temperature. Electroporation 5- Tap the electroporation tube containing the DNA and cells (the cells will settle after being in the tube for a while) and place it in the electroporation tank. 6 . Select the appropriate parameters of the electroporator and start electroporation. 7 - Immediately after electroporation, add 500~900ul of complete medium to the tube and transfer the electroporated cells to a suitable culture vessel (e.g. 6-well plate). Add medium to maintain continued cell growth. 8 . Incubate the cells overnight under suitable growth conditions. If significant cell death is observed, replace the complete medium. Gene expression analysis 9 . If the purpose of the experiment is to stabilize the transfection, skip to step 100. For transient expression, collect the cells 24-72 h after transfection, and select the appropriate method to detect the gene expression, such as using flow cytometry, RNA hybridization, immunization or enzyme assay. If the transfected gene is labeled with a section of fluorescent tag, it can be observed by fluorescence microscopy. 1 0 . Obtaining stably transfected cells: after growing in complete medium for 48~72 h , transfer the cells to suitable medium. Continue to culture the cells under suitable conditions. Examples of operation and parameters 1-Cells and density: IOOul of cell culture medium containing IO6 S V E C (A T C C ) cells. 2-Translocation material: 2f x g p E G F P - N l plasmid D N A (Clontech). 4- Preparation: a- Digest the cells with trypsin-E D T A and wash the cells once with serum-free D M E M medium. b. Collect cells by centrifugation at 250 g for 5 min at room temperature. c. Resuspend cells with IOOmI DMEM medium and mix with Vg plasmid DNA. d. Transfer the mixture into an electroporation tube with an inner diameter of 4m m . e - Place the tube in the electroporation tank of the BTX ECM830 Electroporator and apply a brief voltage of 150 V/cm for 50 ms. For more product details, please visit Aladdin Scientific website.
This step ensures that the cells are healthy and have a high proliferative capacity, which will enhance the tolerance of the cells to electroporation and increase the efficiency of electrotransfection.
For stable transfection, simply transfer a portion of the electroporated cells (1/10 to 1/20 volume) from the tube to the culture vessel.
3 . Electroporation buffer: D M E M cell culture medium.