Hi,
Firstly, I want to say Thank You All to see my questions
I have a fundamental questions to see Fl imaging after DNA transfection.
I did DNA transfection into Neuro2A cell on 6-well. when i see FL image of each cell, FL signal of control seems whole cell body because it is just iRFP protein and FL signal of Target cells seems it is related specific subcellular organelle. (only some spots in the cell)
Before that, I wanted to check transfection efficiency and this is my experimental design.
i) Trasfect cell with DNA plasmid on 6-well (control and target dna plasmid each)
ii) after 1 day, transfer cells on slide glass for confocal imaging
iii) DAPI staining and check (# of transfected cell/# of whole cell) x 100 to check the efficiency
and the questions are
i) do i need to fix the cell to see confocal imaging?
ii) is it okay transfer cell from 6-well to cover glass after cell transfection
If there is better protocol or opinion, Please tell me, it would be very thankful.
Thank You,
Jeongwon Park
It will depend on what kind of microscopes you have access to at your institution. An inverted confocal microscope can be used on glass bottom plates. Alternatively, you can place a sterile round coverslip on the bottom of the wells before culturing your cells (you may need to coat the coverslip depending on your cells behavior) and transfer this to a slide after fixation for imaging.
However, flow cytometry is a much better way of measuring transfection efficiency. You'll need to some basic setup, but the results are much more quantitative than what you will be able to accomplish with microscopy.
We do live cell imaging of cells after transfections which means if you have a fluorescent construct that you are transfecting , you do not need to fix the cells to image. Secondly, you could try and grow and transfect cells on cover slips that you could directly mount on the slides instead of having to transfer. We also use what’s called a mat-tek dish which are glass bottom dishes and transfect in that dish and we are able to directly put it under the microscope and image. So what I mean is I don’t prefer to transfer cells after transfection.
Debadrita Pal , John Hardy Lockhart Thank You for giving me an advice. It would be really helpful!!
However, i have another question for an imaging. Even though i can transfect cell, I'm not sure which timepoint is kinda correct for identifying Transfection efficiency.
Which time point do i need to do imaging (e.g. one day after transfection, or 3 days after transfection)?
When i see the FL signal, it persists until 3days but weakens.
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