: It allows for the quantification of intracellular features, such as the translocation of signals into the nucleus or the internalization of particles. Key Protocol Steps
While IFC shares many preparatory steps with conventional flow cytometry, it requires specific optimizations to ensure high-quality imagery. Imaging Flow Cytometry: Methods and Protocols
: Cells must be in a single-cell suspension to avoid clogging the thin fluidic channels used for imaging. : It allows for the quantification of intracellular
: If internal markers (e.g., nuclear proteins) are the target, cells are fixed with formaldehyde and permeabilized with detergents like Triton X-100. : If internal markers (e
: Non-specific binding is prevented using anti-Fc antibodies. Cells are then stained with fluorophore-conjugated antibodies, viability dyes, or nuclear dyes like DAPI or Hoechst to provide spatial landmarks.
: Systems like the ImageStream use Charge-Coupled Device (CCD) cameras with Time Delay Integration (TDI) to track cells in flow, preventing motion blur and enabling the detection of dim markers.