I am doing the PC12-C6 co-culture system and would like to double stain the cells to observe the cell to cell interactions. I first used the mouse FITC-conjugated anti-beta III tubulin antibody, and then used propidium iodide to stain the cell nuclei. Will the signals from propidium iodide strong enough to eliminate the signals from anti-beta III tubulin antibody?
Hello! The fluorescence spectra of the dyes overlap (493 / 636 nm for PI and 495/519 nm for FITC), you can try changing the fluorescence parameters to separate, but I am not sure if it will work. PI will stain the cell nuclei and tubulin predominantly the cytoplasm, but I doubt you can get good data. You could try splitting up the experiments, for example by adding control groups, if possible.
Georgy Leonov that's only partially correct, while the excitation spectrum of PI is so broad that it overlaps with everything, the emission spectra are very well separated. Using proper filters would allow to detect the two stains independently.
see also my answer to Huang's new question: https://www.researchgate.net/post/Is_it_possible_to_do_the_double_staining_of_anti-beta_tubulin_antibody_and_propidium_iodidePI_staining_for_the_co-culture_of_PC12_and_C6_cells3
Andrea Galli My apologies, in practice I did not try this double staining. Thanks for corrections.
I'm not sure what is the goal of this experiment. Yes, PI and FITC channels will work well enough in fixed cells. However, there is no real reason not to substitute the PI with DAPI, as it is a mere nuclear stain. You don't need nuclear staining in cell-to-cell interaction experiments. Your magnification is too low to see any meaningful cell-to-cell interaction; you need 63x or even 100x oil. You can also try phalloidin dye to visualize the cytoskeleton combined with cell membrane dye such as Concanavalin A. You also may want to add some cell markers to demonstrate that interacting cells are actually PC12 and C6 or label them with the tracers. The ideal method for cell-to-cell interaction is Scanning electron microscopy if such a technique is available to you.
Of course, I think that the amount of PI used was too high, which cause.d the first color to be covered.
Hello Yilun,
Propidium iodide utilised for nucleus/cell cycle labelling can be multiplexed with Fitc/Alexa fluor 488 conjugated anti-beta tublin labelling for flow cytometry. I assume they could be discriminated in microscopy, if the intensity of the tubulin staining is strong. Using DAPI or Hoechst to label nuclei may improve your resolution - any energy transfer DAPI -> FITC working in your favour for excitation. Or a Far Red e.g. Vybrant DC Ruby or FxCycle dye may give better green/red differentiation, by separating the individual dye excitation/emission intensities.
Best wishes
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