i want to do immunofluorescence on sorted cells. These cells are sorted by anti-PE/anti-dextran by flow cytometry. I am not sure if this may cause any background for immunofluorescence and also which secondary antibody i should use? i have 488, Cy3, Cy5 now. Thank you so much
PE is fluorescent in the red-orange range. It is excited from 488 to 561 laser lines. Read here: https://www.thermofisher.com/il/en/home/life-science/cell-analysis/fluorophores/r-phycoerythrin.html
Far-red secondaries may be a good option but trying out is the best way to know.
Try different laser lines without the application of secondary antibodies to see if there is any background fluorescence. Choose the one with minimum fluorescence and apply appropriate secondary antibodies.
Hi Tianyi,
Yes, you can do immunofluorescence on PE-sorted cells.
PE is great for flow cytometry but not very stable for microscopy. Check unstained cells straight after sorting and avoid using Cy3-conjugated secondary antibodies.
Most likely Cy5 will be your choice.
Hi, you can certainly do IF after sorting. As other suggested, check first unstained cells and find the best Fluorophore for your experiment. AlexaFluor 647 or 680 or 405 should be ok. Good luck
Cy5 should work for regular fluorescent microscopy. For confocal microscope you have much more options. You can check spectral viewer for PE and Cy5 or other dyes combinations (link below). As other suggested just look at your unstained cells and single PE- stained cells under different filters available to see if there is any PE spillover into other channels and check the autofluorescence in all the channels available on your microscope. As a rule the dimmer the dye (far red) the less autofluorescence it produces. Dead/apoptotic cells can give huge background in any fluorescent channel.
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html?gclid=EAIaIQobChMI1YOJzcqe5QIVA2yGCh1NiwkOEAAYASAAEgKv1_D_BwE&ef_id=EAIaIQobChMI1YOJzcqe5QIVA2yGCh1NiwkOEAAYASAAEgKv1_D_BwE:G:s&s_kwcid=AL!3652!3!231922595944!e!!g!!spectral%20viewer
Your cells are sorted carrying the PE label, hence all cells are PE positive. You need to use a different label for further staining. Alexa 488 could work. CY3 has a similar excitation and emission to PE...so should not work. Cy5 has a longer emission wavelength to PE...so could work.
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