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Fluorescence Activated Cell Sorting (FACS)

Simplified principle of FACS
Simplified principle of FACS. Graphic made with Biorender.
Fluorescence activated cell sorting (FACS) is a specialized method of flow cytometry that uses fluorescent markers to target and isolate cell groups based on cell surface markers. This method works in the fact that antigenic ligands, like proteins and carbohydrates, give each cell a unique surface phenotype, which can be used as specific targets.

FACS provides researchers the ability to separate cells based on physical characteristics like size, granularity, and cytokine expression. This cell sorting technique has highly dynamic capabilities, may allow for high throughput testing, and is commonly used in hematopoiesis, oncology, and stem cell biology research. FACS is a flow cytometric procedure that provides the ability to also measure and characterize multiple cell generations within a sample. In this way FACS can simultaneously gather expression data and sort cell samples by the required input variables.



Differences between Flow Cytometry & FACS

Both flow cytometry and FACS techniques use fluorescence and other properties to highlight the differences in cell surface or intracellular components of the different cell types in a mixture. Both techniques acquire fluorescent, forward-scatter, and side-scatter data. Flow cytometry and FACS can both be used in a wide range of applications in medicine, immunology, molecular biology, pathology, and genetics.

However, there are several differences between them:

Basis of differentiationFlow CytometryFACS
DefinitionFlow cytometry is an analytical cell biology technique used to identify and study the characteristics of cells in a heterogeneous mixture. It uses differential light scattering properties unique to each cell type in the mixture to determine the number and size of cells and nucleic acid content of the cells.FACS (fluorescence-activated cell sorting) is a specialized type of flow cytometry that facilitates the sorting out cells in a heterogeneous mixture into two or more types. It uses fluorescent-labeled antibodies to specifically identify components of different cell types
Type of techniqueThis is an analytical cell biology technique.This is a specialized type of flow cytometry.
Sampling method usedThe process uses the differential light-scattering properties of cells to collect the necessary data.The process uses highly specific antibodies tagged with fluorescent dyes to distinguish between cell types.
Analysis methodA sensor is used to acquire data.An electromagnet is used to sort the sample.
SequenceFlow cytometry follows FACS.FACS is the first step of analyzing a heterogeneous cell mixture. It is followed by flow cytometry.
FunctionThis technique measures certain cell characteristics such as the number, size, and nucleic acid content of cells.This technique separates cells from a heterogeneous mixture into appropriate subpopulations.



DNA profile in growing and nocodazole treated Jurkat cells
DNA profile in growing and nocodazole treated Jurkat cells. Jurkat cells were treated without (A) or with Nocodazole (B) for 24 hours, then incubated with Nuclear Green™ LCS1 for 30 minutes. The fluorescence intensity of Nuclear Green™ LCS1 was measured with an ACEA NovoCyte flow cytometer in the FITC channel. In growing Jurkat cells (A), nuclear staining with Nuclear Green™ LCS1 shows G1, S, and G2 phases. In Nocodazole treated G2 arrested cells (B), the frequency of G2 cells increased dramatically, while G1 and S phase-frequency decreased significantly.
FACS offers a number of applications in the field of research and diagnostics. In immunophenotyping, FACS can identify and quantify multiple populations of cells in a single heterogeneous sample for peripheral blood, bone marrow, and even lymph material. Commonly, FACS is used in a hematological setting in which specific cancer subtypes may be diagnosed. FACS may also be used for the purpose of cell cycle analysis, where cells can be analyzed and measured in all four distinct phases.

Secondary cell-based assays can also be used to further assist with the determination of cell anomalies using certain fluorophores, a common practice throughout single-cell genomics. FACS may additionally be used to assess cell viability and proliferation, commonly performed by labeling resting cells with a membrane fluorescent dye like carboxyfluorescein succinimidyl ester (CFSE). This works by the principle of mitosis; as cells grow and divide, half of the dye is passed to each daughter. By measuring the reduction in fluorescent signal, cellular activation and proliferation can be determined.

The extent of apoptosis and necrosis within a sample can also be identified and distinguished, which may be helpful in determining morphological, biochemical, or molecular changes that occur over time. FACS has also been used to assess membrane potential and interpreting ion flux within a sample. This technique provides the capability of detecting the flux of calcium ions that are drawn into a cell, which thereby measures the activation of associated signal transduction pathways. FACS has a number of other applications, including:
  1. Assessing pH changes
  2. Evaluating redox state or oxidative metabolism
  3. Analyzing chromatin structure and gene expression
  4. Gauging total protein expression, lipid population, or enzyme activity
  5. Providing light on the extent of DNA replication and/or degradation

Methodology and Principles

Detection of Jurkat cell viability by Cell Meter™ fixable viability dye
Detection of Jurkat cell viability by Cell Meter™ fixable viability dye. Jurkat cells were treated and stained with Cell Meter™ VX450 and then fixed in 3.7% formaldehyde and analyzed by flow cytometry. The dead cell population (Blue peak) is easily distinguished from the live cell population (Red peak) with AmCyan channel, and nearly identical results were obtained before and after fixation.
 The methodology for a FACS experiment is straightforward.
  1. Cells in suspension are prepared with a stain of fluorescently-targeted monoclonal antibodies (mAb) that recognize specific surface markers within the desired cell population.
  2. Predetermined fluorescent parameters of the cells of interest can be input into a flow cytometer, and sorting parameters can be adjusted depending on the desired output purity and yield of the sample.
  3. The suspension is passed through the instrument as a stream of droplets, each containing a single cell, in front of a laser.
  4. The flow cytometer applies a charge to each droplet and an electrostatic deflection system facilitates the assembly of the charged cells into appropriate collection tubes for analysis and quantification.
Note: In FACS technology, the success of staining, and therefore sorting, depends heavily on the choice of markers and mAbs used. Additionally, the FACS process is inherently slow in that a low stream flow rate must be maintained to accurately identify cells.

FACS offers a number of advantages over other methods of cell sorting, and is especially useful for detecting very low levels of protein expression. FACS is the only available purification technique that utilizes size, granularity, and marker detection via fluorescent targeting of intracellular proteins. FACS is largely useful if separation based on differential marker density is required, and the technique provides the ability to negatively select unstained cells, if necessary.

Flow cytometry, in general, requires an adequate number of cells in the starting material, commonly around 1 million, as staining and washing procedures will cause cell loss. It should be stated that the recovery rate of FACS is, on average, between 50-70%, which could pose a disadvantage when working with rare cells. The choice of flow cytometer must also be taken into consideration, as FACS purification requires a strong sorting capacity, with an appropriately coupled software.

Adapted and Integrated FACS Techniques Used in Research

Pairing FACS with other experimental techniques has recently given researchers the ability to better explore human biology. FACS has been used in tandem with targeted analysis of histone modification to better profile primary human leukocytes. This research has helped contribute to knowledge of histone post-translational modification, required for differentiation and maintenance of certain distinct cell types. Some research has also integrated microfluidic devices with FACS to create a miniaturized analytical system that can perform similarly to a flow cytometer. The development of this technology is aimed towards diagnostic applications with the ultimate goal of a low-cost, portable instrument for point of care use.

Other research has focused on making FACS techniques ultra-high-throughput by integrating directed evolution of enzymes and proteins. Directed evolution of binding proteins has provided a novel method of efficiently identifying variants with high affinity and selectivity for mapping specific protein interactions. Furthermore, FACS has been used in research alongside magnetic-activated cell sorting (MACS) to distinguish between undifferentiated human embryonic stem cells (hESCs) from a heterogeneous cell population. This research is essential in hESC-derived cell replacement therapy, as the major risk in this procedure lies in the unknown tumorigenesis potential from undifferentiated hESCs.

Table 1. Common CD markers used for the differentiation of leukocytes by flow cytometry.

Cell Type
Common Human CD Markers
Common Mouse CD Markers
T CellCD3, CD4, CD8CD3, CD4, CD8
B CellCD19, CD20CD45R/B220, CD19, CD 22 (B cell activation marker)
Dendritic CellCD11c, CD123CD11c, CD123
Natural Killer (NK) CellCD56CD335 (NKp46)
Stem Cell/PrecursorCD34 (hematopoietic stem cell only)CD34 (hematopoietic stem cell only)
Monocyte/MacrophageCD14, CD33CD11b/Mac-1, Ly-71 (F4/80)
GranulocyteCD66bCD66b, Gr-1/Ly6G, Ly6C
PlateletCD41, CD61, CD62CD41, CD61 (Integrin Β3), CD9, CD62P (activated platelets)
ErythrocyteCD235aCD235a, Ter-119
Endothelial CellCD146CD146 MECA-32, CD106, CD31, CD62E (activated endothelial cells)
Epithelial CellCD326CD326 (EPCAM1)

Product Ordering Information


Table 2. CytoTell™ and CFSE cell proliferation dyes for flow cytomtery

Product Name
Excitation Source (nm)
Ex (nm)
Em (nm)
Unit Size
Cat No.
CytoTell™ BlueViolet Laser (405 nm)410445500 Tests22251
CytoTell™ Violet 500Violet Laser (405 nm)415499500 Tests22248
CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]Blue Laser (488 nm)49851725 mg22022
ReadiUse™ CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]Blue Laser (488 nm)4985175 x 500 µg22028
CytoTell™ GreenBlue Laser (488 nm)510525500 Tests22253
CytoTell™ UltraGreenBlue Laser (488 nm)492519500 Tests22240
CytoTell™ OrangeGreen Laser (531 nm)541560500 Tests22257
CytoTell™ Red 590Green Laser (531 nm)560574500 Tests22261
CytoTell™ Red 650Red Laser (633/647 nm)626643500 Tests22255
CytoTell™ BlueViolet Laser (405 nm)4104452 x 500 Tests22252
CytoTell™ Violet 500Violet Laser (405 nm)4154991000 Tests22249
CytoTell™ GreenBlue Laser (488 nm)5105252 x 500 Tests22254
CytoTell™ UltraGreenBlue Laser (488 nm)4925192 x 500 Tests22241
CytoTell™ OrangeGreen Laser (531 nm)5415602 x 500 Tests22258
CytoTell™ Red 590Green Laser (531 nm)5605742 x 500 Tests22262
CytoTell™ Red 650Red Laser (633/647 nm)6266432 x 500 Tests22256

Table 3. Cell cycle assays and reagents for flow cytometry.

Unit Size
Cat No.
7-AAD [7-Aminoactinomycin D] *CAS 7240-37-1*1 mg17501
Cell Navigator® CDy6 Mitosis Imaging Kit100 tests22640
Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit *Optimized for 405 nm Violet Laser Excitation*100 tests22842
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Green Fluorescence Optimized for Flow Cytometry*100 tests22841
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Optimized for 405 nm Violet Laser Excitation*100 tests22845
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*100 tests22860
DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *10 mM solution in water*2 mL17507
DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *CAS 28718-90-3*10 mg17510
DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *CAS 28718-90-3*25 mg17513
DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *CAS 28718-90-3*100 mg17511
Hoechst 33258 *CAS 23491-45-4*100 mg17520
Hoechst 33258 *CAS 23491-45-4*1 g17523
Hoechst 33258 *20 mM solution in water*5 mL17525
Hoechst 33342 *Ultrapure Grade*100 mg17530
Hoechst 33342 *Ultrapure Grade*1 g17533
Hoechst 33342 *20 mM solution in water*5 mL17535
Propidium iodide *CAS 25535-16-4*25 mg17515
Propidium iodide *CAS 25535-16-4*5 g17516
Propidium iodide *10 mM aqueous solution*1 mL17517
Acridine orange 100 mg17502
Acridine orange *10 mg/mL solution in water*10 mL17503

Table 4. Properties of cytocalcein AM viability dyes optimized for flow cytometry

Mol. Wt
Ex/Em (nm)
Unit Size
Cat No.
CytoCalcein™ Violet 450 *Excited at 405 nm*∼600406/445 nm1 mg22012
CytoCalcein™ Violet 500 *Excited at 405 nm*517.93420/505 nm1 mg22013

Table 5. Available PE and APC tandem dyes for multicolor flow cytometry.

Tandem Dye
Ex (nm)
Em (nm)
Stoke's Shift (nm)
Unit Size
Cat No.
PE-Texas Red Tandem495, 565615120, 501 mg2619
PE-Cy5 Tandem495, 565666171, 1011 mg2610
PE-Cy5.5 Tandem495, 565671176, 1081 mg2613
PE-Cy7 Tandem495, 565778283, 2131 mg2616
PE-iFluor® 594 Tandem495, 565606111, 411 mg2600
PE-iFluor® 610 Tandem495, 566628133, 621 mg2700
PE-iFluor® 647495, 569666171, 971 mg2702
PE-iFluor® 660 Tandem495, 565678183, 1131 mg2602
PE-iFluor® 700 Tandem495, 565708213, 1431 mg2614
PE-iFluor® 710 Tandem495, 565739244, 1741 mg2615
PE-iFluor® 750495, 566778283, 2121 mg2704
PE-iFluor® 780 2617
APC-Cy5.5 Tandem651700491 mg2622
APC-Cy7 Tandem6517791281 mg2625
APC-iFluor®A7 Tandem651782-1 mg2632
APC-iFluor® 700 Tandem651710591 mg2623
APC-iFluor® 710 Tandem 651740 1 mg2631
APC-iFluor® 750 Tandem6517781271 mg2626
APC-iFluor® 800 Tandem6518201691 mg2630
APC-XFD700 Tandem651719681 mg2624
APC-XFD750 Tandem6517761251 mg2627
PerCP-Cy5.5 Tandem4896791901 mg2650
ReadiUse™ Preactivated PE-Texas Red Tandem495, 565615120, 501 mg2583
ReadiUse™ Preactivated PE-Cy5 Tandem495, 565666171, 1011 mg2580
ReadiUse™ Preactivated PE-Cy5.5 Tandem495, 565671176, 1081 mg2581
ReadiUse™ Preactivated PE-Cy7 Tandem495, 565778283, 2131 mg2582
ReadiUse™ Preactivated PE-iFluor® 594 Tandem495, 565604109, 391 mg2584
ReadiUse™ Preactivated PE-iFluor® 647 Tandem495, 565666171, 1011 mg2577
ReadiUse™ Preactivated PE-iFluor® 660 Tandem495, 565678183, 1131 mg2579
ReadiUse™ Preactivated PE-iFluor® 700 Tandem495, 565722227, 1571 mg2585
ReadiUse™ Preactivated PE-iFluor® 750 Tandem495, 565778283, 2131 mg2578
ReadiUse™ Preactivated APC-Cy5.5 Tandem651700491 mg2586
ReadiUse™ Preactivated APC-Cy7 Tandem6517791281 mg2587
ReadiUse™ Preactivated APC-iFluor® 700 Tandem651710591 mg2570
ReadiUse™ Preactivated APC-iFluor® 750 Tandem6517911401 mg2571
ReadiUse™ Preactivated APC-iFluor® 800 Tandem6518201691 mg2572
ReadiUse™ Preactivated PerCP-Cy5.5 Tandem4896791901 mg2595

Table 6. Fixable viability dyes for live/dead cell analysis during flow cytometry

Ex (nm)
Em (nm)
Unit Size
Cat No.
Cell Meter™ VX450 fixable viability dye406445200 Tests22540
Cell Meter™ VX500 fixable viability dye433498200 Tests22542
Cell Meter™ BX520 fixable viability dye491516200 Tests22510
Cell Meter™ BX590 fixable viability dye492579200 Tests22514
Cell Meter™ BX650 fixable viability dye518654200 Tests22520
Cell Meter™ RX660 fixable viability dye649664200 Tests22530
Cell Meter™ RX700 fixable viability dye690713200 Tests22532
Cell Meter™ RX780 fixable viability dye629767200 Tests22536
Cell Meter™ IX830 fixable viability dye811822200 Tests22529
ReadiView™ Green/Red Viability Stain 100 Tests22762

Table 7. Live or Dead™ Fixable Dead Cell Staining kits for live/dead cell analysis during flow cytometry

Laser (nm)
Ex/Em (nm)
Unit Size
Cat No.
Live or Dead™ Fixable Dead Cell Staining Kit *Blue Fluorescence*UV (350 nm)353/442YesYes200 Tests22600
Live or Dead™ Fixable Dead Cell Staining Kit *Blue Fluorescence with 405 nm Excitation*Violet (405 nm)410/450YesYes200 Tests22500
Live or Dead™ Fixable Dead Cell Staining Kit *Green Fluorescence with 405 nm Excitation*Violet (405 nm)408/512YesYes200 Tests22501
Live or Dead™ Fixable Dead Cell Staining Kit *Green Fluorescence*Blue (488 nm)498/521YesYes200 Tests22601
Live or Dead™ Fixable Dead Cell Staining Kit *Orange Fluorescence with 405 nm Excitation*Violet (405 nm)398/550YesYes200 Tests22502
Live or Dead™ Fixable Dead Cell Staining Kit *Orange Fluorescence*Green/Yellow (532/561 nm)547/573YesYes200 Tests22602
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence*Yellow (561 nm)583/603YesYes200 Tests22603
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*Blue/Green (488/532 nm)523/617YesYes200 Tests22599
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*Red (635 nm)649/660YesYes200 Tests22604
Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*Red (635 nm)749/775YesYes200 Tests22605



Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)
Development of a microfluidic device for fluorescence activated cell sorting
Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts
Understanding the Relationship Between FACS and Flow Cytometry
Coupling Fluorescence-Activated Cell Sorting and Targeted Analysis of Histone Modification Profiles in Primary Human Leukocytes
Development of a microfluidic device for fluorescence activated cell sorting
Separation of SSEA-4 and TRA-1-60 Labelled Undifferentiated Human Embryonic Stem Cells from A Heterogeneous Cell Population Using Magnetic-Activated Cell Sorting (MACS) and Fluorescence-Activated Cell Sorting (FACS)