: Cells must be in a single-cell suspension to avoid clogging the thin fluidic channels used for imaging.
: 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.
: 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 Imaging Flow Cytometry: Methods and Protocols
: 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.
: Researchers must set specific software parameters, such as magnification and laser power, to maximize the resolution of the captured images. Common Applications : Cells must be in a single-cell suspension
: Beyond fluorescence, IFC provides data on cell size, shape, circularity, and internal complexity.
IFC provides a "visual verification" for every data point, allowing researchers to see exactly what their gated populations look like. Key Protocol Steps : Non-specific binding is prevented
: If internal markers (e.g., nuclear proteins) are the target, cells are fixed with formaldehyde and permeabilized with detergents like Triton X-100.