Hello,
I am asking if is possible to use flow cytometry cells for an IF. I am new to doing IF. Say I stain my cells for flow cytometry with three colors: fitc, pe-texas red, and dapi. I do this by fixing my cells, washing then staining with primary. Then a final two washes. Is it possible or feasible to take the cell suspension for the last wash and spin them down to attach for a coverslip to use for IF?
Dear Kendell
First, the cells might not adhere to the coverslip due to other membrane changes.
Second, you should know that the sensitivity of flow cytometry is often higher than that of immunofluorescence, and the desired results might not be obtained using a microscope.
Third, if the goal is to study the precise localization of certain components within the cell using a confocal microscope, this may not be achievable due to morphological changes in the cells caused by the treatments applied.
Dear Kendell,
Adding to the above answer, you cannot directly transfer cells stained for one technique to the other without first modifying the staining protocol to suit the specific needs of the target technique. While flow cytometry and IF share some similarities, they will require specific consideration to ensure optimal results.
While many fluorophores are suitable for both flow cytometry and IF, factors like brightness, photostability, signal amplification needs, and instrument capabilities should be considered when selecting a fluorophore for a specific experiment.
Flow cytometry often benefits from very bright dyes, even if they are not highly photostable (for instance, phycobilins, APC and PE). However, in IF, where signal is often amplified through secondary antibodies, photostability becomes crucial.
Another point which I would like to mention between the two techniques is that flow cytometry can detect antigens even at relatively low levels, often relying on direct staining with conjugated primary antibodies. However, IF may require indirect staining (unlabeled primary + fluorescent secondary) for increased signal.
The fixatives which is used in flow cytometry can interfere with the effectiveness of immunofluorescence permeabilization methods. For instance, formaldehyde cross-links proteins, which can block access to intracellular targets by antibodies. This means that a separate permeabilization step is necessary to allow probes to access intracellular structures. Also, most of the times, flow cytometry staining protocols often do not include permeabilization steps, while immunofluorescence requires specific permeabilization protocols to allow antibodies to access intracellular targets.
Best,