Flow Cytometry and FACS

Core Concept

Flow cytometry is a technique that rapidly measures the properties of single cells as they stream in single file past a laser, one by one, thousands per second. For each cell it records forward scatter (FSC) — roughly proportional to cell size — side scatter (SSC) — related to internal granularity/complexity — and fluorescence from dyes or antibody-conjugated fluorophores that report specific molecules. Fluorescence-activated cell sorting (FACS) is a specific extension of flow cytometry that, after measuring, also physically sorts cells into separate tubes by deflecting droplets in an electric field. The key distinction: flow cytometry = analysis/measurement; FACS = sorting — it is a subset of flow cytometry, not a synonym for all flow cytometry.

Key Components

• Fluidics (sheath fluid): a sheath of fluid focuses cells into a single-file stream (hydrodynamic focusing) so each cell passes the laser individually.
• Laser(s): excite fluorophores; multiple lasers excite different dyes.
• Forward scatter (FSC): light scattered at small angles along the beam axis — increases with cell size.
• Side scatter (SSC): light scattered at ~90° — increases with internal complexity (granules, lobed nuclei, vesicles).
• Fluorescence detectors: photomultiplier tubes behind band-pass filters read each fluorophore's emission, quantifying how much of a labeled molecule is on/in each cell.
• Sorting hardware (FACS only): the stream is vibrated to break into droplets; droplets containing a cell of interest are electrically charged and deflected by charged plates into collection tubes.

Mechanism / How It Works

1. Single-file stream. Cells in suspension are injected into a faster-flowing sheath fluid, which hydrodynamically focuses them into a narrow, single-cell stream.
2. Interrogation. Each cell crosses the laser beam. FSC measures size; SSC measures granularity; each fluorophore's emission is split by optics and measured by a dedicated detector.
3. Data recording. Each cell becomes one dot on a scatter plot (e.g., FSC vs. SSC, or fluorescence vs. SSC). A population of similar cells forms a cluster ("gating" selects it). Multiparameter (multichannel) data lets one cell be characterized by many markers at once.
4. Sorting (FACS). After measurement, the stream is vibrated so it breaks into droplets, most containing one cell. If a droplet's cell matches the sort criteria, the droplet is given an electric charge at the moment it separates; charged deflection plates then steer it into a collection tube. Unwanted droplets pass to waste. Cells can be sorted sterilely and kept alive for culture.
5. Output. Flow cytometry yields population statistics (percentages, intensities); FACS yields pure, collected subpopulations for downstream experiments (culture, sequencing, transplant).

Energy and Directionality

The measurement uses laser light (optical energy), not cellular ATP; fluorescence is the passive emission described in the fluorescence note. The sorting step uses an electric field to deflect charged droplets — electrostatic energy, not the cell's metabolism. Live cells can be sorted because the process is fast and gentle enough; they retain their own ATP-driven metabolism for downstream culture.

Technique (How It Is Done)

• Label cells: fluorescent antibodies against surface markers (e.g., CD markers on leukocytes), viability dyes, DNA dyes (propidium iodide, DAPI), or fluorescent proteins.
• Run controls: unstained cells (set baseline/autofluorescence), single-color controls (compensation to correct spectral overlap between dyes), and isotype controls.
• Gate and analyze: define populations on FSC/SSC and fluorescence plots; report percentages and intensities.
• Sort (FACS): set sort gates, collect into tubes/plates; verify purity by re-running the sorted fraction.
• Applications: immunophenotyping (identifying lymphocyte subtypes by CD markers); cell cycle (DNA content by dye intensity → G1 vs. S vs. G2/M); apoptosis (Annexin V binds exposed phosphatidylserine); cell viability (dye exclusion).

How it works

1. Single-file stream. Cells in suspension are injected into a faster-flowing sheath fluid, which hydrodynamically focuses them into a narrow, single-cell stream.
2. Interrogation. Each cell crosses the laser beam. FSC measures size; SSC measures granularity; each fluorophore's emission is split by optics and measured by a dedicated detector.
3. Data recording. Each cell becomes one dot on a scatter plot (e.g., FSC vs. SSC, or fluorescence vs. SSC). A population of similar cells forms a cluster ("gating" selects it). Multiparameter (multichannel) data lets one cell be characterized by many markers at once.
4. Sorting (FACS). After measurement, the stream is vibrated so it breaks into droplets, most containing one cell. If a droplet's cell matches the sort criteria, the droplet is given an electric charge at the moment it separates; charged deflection plates then steer it into a collection tube. Unwanted droplets pass to waste. Cells can be sorted sterilely and kept alive for culture.
5. Output. Flow cytometry yields population statistics (percentages, intensities); FACS yields pure, collected subpopulations for downstream experiments (culture, sequencing, transplant).

Common confusions

• "FACS and flow cytometry are the same thing." — Wrong. Flow cytometry measures; FACS measures and sorts. All FACS is flow cytometry, but not all flow cytometry is FACS.
• "FSC measures granularity and SSC measures size." — Reversed. FSC ≈ size; SSC ≈ granularity/complexity.
• "Fluorescence intensity tells you the exact number of molecules." — It is proportional to the amount of labeled molecule, but requires standards/calibration for absolute quantification.
• "Cells must be dead to be analyzed." — No. Live cells are routinely analyzed and even sorted sterilely for further culture.
• "One dot on the plot = one molecule." — One dot = one cell; the position reflects that cell's scatter/fluorescence values.

Quick review

• Single cells stream past a laser (hydrodynamic focusing); thousands/second.
• FSC = size; SSC = granularity; fluorescence = specific labeled molecules.
• Flow cytometry = measurement; FACS = measurement + sorting (charged droplets).
• Populations form clusters on scatter/fluorescence plots (gating).
• Uses: immunophenotyping (CD), cell cycle (DNA content), apoptosis (Annexin V), viability.
• Compensation corrects spectral overlap between fluorophores.