Hi, i am now going to label one population(A), and doing the co-culture with another cells(B), after some treatment(about 3days), i want to separate A cells by FACS, is there somebody have experience with some methods, like cell-lable dye, or GFP transfection? I wonder for this purpose, whether dye or transfection is reliable and which method is easy to handle? Thanks.
Best,
Ken
Dear Ken
It will depend on the proliferation speed of your cells. In case the population you seek to sort out is proliferative, such as cell lines, it might be advised to stably transfect your cells with a fluorescent protein such as GFP. Take note that stable transfection is advised as you want to co-incubate with another cell type for 3 days, and you will probably transfect the cells one day in advance. This will add up to 4 days, in which your expression level will be way too low to do accurate cell sorting due to a low noise/signal ratio. Stable transfection, however, will require a longer preparation as you need to achieve this prior to co-cultivating.
An alternative method could be to stain your cells with CFSE. CFSE is a dye often used in immunology, as it stains your cells and is passed to the daugher cells during cell division. Take note that high proliferative cells will therefore also lose intensity over time. Additionally, CFSE is quite light sensitive so try not to expose it too much.
In any case, you could try both approaches in a single cell type set-up, and determine your recovery rate of your stained cells from unstained cells in a flow cytometry experiment in which you use e.g. 50% stained vs 50% unstained cells.
Good luck
Philippe
Hello Kecheng,
Selection of GFP label or some dye staining depends on for how much time you are gonna culture and observe cells after labelling. For 3 days, even CFSE will work fine and you can easily differentiate labelled population from unlabelled one on FACS (in this case you will be saved from lots of trouble involoving transfection). If you want to go for long term experiments, go with GFP labelling. In both cases, FACS can easily differentiate the labelled population.
Hope it helps !
PKH26 (red) vs PKH67 (green) are simple to use dyes and the cells retain fluorescence upto a week time (in my experience). This should help to solve your problem. See my paper for cat #s
https://www.researchgate.net/publication/291526211_Endocytosis_and_serpentine_filopodia_drive_blebbishield-mediated_resurrection_of_apoptotic_cancer_stem_cells
Article Endocytosis and serpentine filopodia drive blebbishield-medi...
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