Key Takeaways

• FACS isolates individual cells with a purity approaching 99.99%.
• It sorts cells by size, granularity, surface markers and fluorescence intensity.
• Cells are labelled with fluorochrome-conjugated antibodies such as FITC, PE and APC.
• Charged droplets containing single cells are deflected into collection tubes by electric plates.
• Applications span cancer research, immunology, stem cell biology and clinical diagnostics.
• FACS is a sorting-capable subset of flow cytometry — all FACS is flow cytometry, but not all flow cytometry sorts.

Contents

• What is FACS?
• How Does FACS Work?
• The FACS Protocol
• FACS vs Flow Cytometry
• Applications of FACS
• Frequently Asked Questions

Reagents for FACS & Flow Cytometry

From fluorochrome-conjugated marker antibodies to viability dyes and fixation buffers, these validated reagents support every stage of a cell-sorting experiment.

CD3

CD3 Monoclonal Antibody (FITC)

FITCHuman

A pan-T-cell marker in a green fluorochrome for the first sorting channel.

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CD4

CD4 Monoclonal Antibody (PE-Cy5)

PE-Cy5Mouse

Identifies helper T cells for multi-colour sorting panels.

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CD8

CD8 Monoclonal Antibody (APC)

APCFlow

A far-red conjugate for resolving cytotoxic T cells alongside CD3 and CD4.

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7-AAD

7-AAD Viability Staining Solution

Viability DyeFlow

Excludes dead cells so only viable events are sorted.

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Fix/Perm

Intracellular Fix/Perm Buffer Kit

Buffer KitIntracellular

Fixes and permeabilises cells to stain intracellular targets.

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Annexin V

Annexin V-FITC/PI Apoptosis Kit

Apoptosis KitFITC · PI

Distinguishes live, apoptotic and necrotic cells during sorting.

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What is FACS?

Fluorescence-activated cell sorting is a technique for isolating individual cells from a heterogeneous sample and analysing their properties one cell at a time. It is a specialised, sorting-capable form of flow cytometry, and it is revolutionising cell biology by allowing researchers to purify defined cell populations with an accuracy of up to 99.99% — making it ideal for any application where cell purity matters.

The power of FACS lies in combining measurement and physical separation. As each cell passes a laser, the instrument records its size, internal complexity and the fluorescence of any bound markers, then diverts that individual cell into the correct collection tube — all in a fraction of a second.

How Does FACS Work?

A FACS instrument works by carrying cells single-file in a fast-moving fluid stream past one or more laser beams. As each cell crosses a laser, forward-scatter reports its size and side-scatter its internal granularity, while detectors capture the light emitted by fluorochrome-labelled markers on or inside the cell.

The stream is then broken into thousands of tiny droplets, each ideally containing a single cell. Droplets carrying a cell of interest are given an electric charge, and as they fall between two deflection plates, positively charged droplets are pulled toward the negative plate and negatively charged droplets toward the positive plate. The sorted cells are collected in separate tubes, while unwanted droplets pass straight into waste.

The FACS Protocol

FACS is a complex procedure requiring trained personnel and specialised equipment, but the core workflow is consistent. First, the cells are labelled with fluorescent dyes — usually antibodies conjugated to fluorochromes such as FITC (a green dye excited by blue light) or PE (a red dye excited by green light) that bind specific cell-surface markers.

The cells are incubated with the antibodies for 30 to 60 minutes, then washed and resuspended in a sorting buffer containing salts and proteins that keep the cells healthy during the run. A viability dye such as 7-AAD is often added to exclude dead cells. The prepared sample is then loaded onto the sorter, gated on the populations of interest, and sorted into collection tubes for downstream culture or analysis.

FACS vs Flow Cytometry

The two terms are often used interchangeably, but there is a key distinction. Flow cytometry is the broad technique of measuring the properties of cells in a fluid stream; it analyses cells but does not necessarily separate them. FACS adds the sorting capability — it not only measures each cell but also physically recovers defined populations for further use. In short, all FACS is flow cytometry, but only sorting-capable instruments perform FACS.

Applications of FACS

The ability to purify defined cell populations makes FACS indispensable across the life sciences.

Cancer research

FACS isolates tumour and immune cell subsets from complex tissues, enabling the study of cancer heterogeneity and the immune response to tumours.

Immunology

Researchers routinely sort T-cell, B-cell and other leukocyte subsets — using markers such as CD3, CD4 and CD8 — to dissect immune function.

Stem cell research

FACS purifies rare stem and progenitor populations from marrow or tissue based on their unique marker profiles.

Developmental biology

Sorting cells by lineage marker at successive stages reveals how cell populations arise and differentiate during development.