FLUORESCENCE ACTIVATED
CELL SORTING
What is Flow Cytometry?
• Cytometry refers to the measurement of physical/chemical
characteristics of cells or other biological particles.
• Flow Cytometry is the process whereby such measurements
are made upon cells/particles as they pass through a
measuring apparatus (hopefully in single file) suspended in a
fluid stream.
1968, Wolfgang Gohde from the University of Munster
(Patent No. DE1815352)
named ‘pulse cytophotometry”
1978, the name was changed to ‘flow cytometry’
• Flow Sorting (Flow Cytometric Cell Sorting) extends flow
cytometry with the additional capacity to divert and collect
cells exhibiting an identifiable set of characteristics either
mechanically or by electrical means (Flow Cytometric
Analysis).
• FACS - Fluorescence Activated Cell Sorting? FACS is
a trademark of Becton Dickinson Immunocytometry
Systems (BDIS).
Fluorescence activated cell sorting (FACS) of live cells
separates a population of cells into subpopulations based
on fluorescent labeling.
Sorting involves more complex mechanisms in the flow
cytometer than a non-sorting analysis.
Cells stained using fluorophore-conjugated antibodies
can be separated from one another depending on which
fluorophore they have been stained with.
For example, a cell expressing one cell marker may be
detected using an FITC-conjugated antibody that
recognizes the marker, and another cell type expressing
a different marker could be detected using a PE-
conjugated antibody specific for that marker. This is the
basic task of flow cytometry.
Cell Sorters (FACS – Fluorescence Activated Cell Sorter)
Fluidic System
Air Filter
Air Pump
Flow Cell
Sample
Sheath Regulator
Regulator
Sample
Sheath Filter SIT; sample injection tube
Sheath Tank Waste Tank
Optics System
488nm LASER 1
635nm LASER 2
Cell Sorting
1. Individual cells are “interrogated” by the laser as in a
normal flow cytometer.
2. The machine is set up so that each individual cell then
enters a single droplet as it leaves the nozzle tip. This
drop is given an electronic charge, depending on the
fluorescence of the cell inside the drop.
3. Deflection plates attract or repel the cells accordingly
into collection tubes.
3. For example: A single FITC stained cell in a single droplet would
be given a positive charge and be attracted to the right. Collection
tubes to the right would collect all the positively charged FITC
stained cell droplets.
A single PE stained cell in a single droplet would be given a
negative charge and be attracted to the left. Collection tubes to
the left would collect all the negatively charged PE stained cell
droplets.
4. Sorted cell populations are then analyzed to ensure successful cell
sorting.
5. Sorted cells can then be cultured.
Fluorescein isothiocyanate (FITC) is a derivative
of fluorescein used in wide-ranging applications including flow
cytometry. FITC is the original fluorescein
molecule functionalized with an isothiocyanate reactive group (-
N=C=S), replacing a hydrogen atom on the bottom ring of the
structure.
Phycoerythrin (PE) is one of the most commonly-used
fluorescent dyes for FACS analysis. PE is a large protein
(approximate molecular weight 240 kd) containing 25 fluors.
Typically, only one PE molecule is conjugated to an antibody.
Adapted from ComputCyte
Adapted from ComputCyte
