Flow Cytometry

Introduction

Flow cytometry is a widely used method for the separation, classification and quantification of cells. Complex computerised instruments are used to pass a mono-cellular stream of cells, platelets or other microscopic particulate elements through a beam of laser light. The cells are categorised first by size and then computer analysed to sort the mixture of cellular elements into cell type by size. In addition, antibodies conjugated to fluorescent dyes and/or fluorescent intercalating agents can be used to mark specific cell components. Each cell will display an appropriate fluorescent light emission, consistent with the total component presence in the cell. This emission is counted. Tabulation of counted data, in conjunction with size analysis, enables determination of relative percentages of each specific cellular subset for which antibody conjugates are utilised, even when the size of the cell is identical to other subset species. This provides a simultaneous multi-parameter analysis of single cells for the characterisation and definition of different cell types in heterogeneous cell populations.  The staining procedure involves making single-cell suspensions from cell culture or tissue samples. The cells are then incubated in tubes or microtiter plates with or without fluorescently-labelled antibodies and analysed using a flow cytometer.

The Flow Cytometer

The process involves the measurement of scattered light and fluorescence, and is dependent on dynamic fluidics to pass cell suspensions through the flow cytometer.  When an individual cell suspension is run through the cytometer, sheath fluid is used to hydro-dynamically focus the cell suspension through a small nozzle. The fluid transports the cells past a laser light one cell at a time. As the cells pass through the laser beam, any light scattering from the cells or particles is detected. This is detected as forward scatter (FS) or side scatter (SS) which correlates to cell size and granularity respectively. As a result, cell populations are frequently distinguished based on differences in their size and granularity. In addition, fluorescence detectors measure the fluorescence emitted from positively stained cells or particles, which can be used to separate cells based on protein expression.

Forward and side scattered light, and fluorescence from stained cells are split into defined wavelengths and directed by a set of filters and mirrors within the flow cytometer. The fluorescent light is filtered so that each sensor only detects fluorescent emission at a specific wavelength. These sensors are called photomultiplying tubes (PMTs). For example, a PMT channel set-up for the detection of FITC (fluorescein isothiocyanate) will only detect light emitted from FITC or fluorophores emitted at a wavelength of around 519 nm.  As a result, the fluorophores only emit light when excited by a laser with the corresponding excitation wavelength. This can be used to detect fluorescently stained cells or particles individually.

Flow Cytometry Data

Each individual PMT measures fluorophore emission when the fluorescing cells are passed through the laser beam. This emission is detected by the PMT and converted to a voltage pulse, also known as an event. As the fluorescently labelled cells pass through the beam, the intensity of the voltage measured increases. The pulse of voltage is completed as each fluorescing cell travels through the laser beam, which gives a final pulse height and pulse area which can be measured by the flow cytometer.

 

The voltage pulse area correlates directly to the fluorescence intensity of an event. As a result, these events can be assigned to channels based on pulse intensity (pulse area) whereby a higher fluorescence intensity results in the allocation of a higher channel number for an event. This can be used to assess cell populations that are positive and negative for a particular protein of interest. A negative result would be shown as a large number of events at low fluorescence intensities whereas a positive result would be shown as lots of events at high fluorescence intensities.  


The results of flow cytometry are also often displayed as a dot plot. A dot plot can be used to separate cells into different populations based on size (forward scatter), granularity (side scatter) and/or fluorescent labelling (fluorescent emission). This allows for the separation of different cell types into distinct clusters of different cell types such as monocytes and lymphocytes [left]. Furthermore, dot plots can be used to separate and analyse normal (viable) cells, from apoptotic cells and dead cells based on cell labelling with fluorophore-conjugated Annexin V (e.g. Annexin V-FITC) and a DNA stain such as propidium iodide (PI) [right].


Fluorophores

The ability of a given antibody or dye to resolve a positive signal from a negative signal often depends on which fluorophore is used. For example, a highly expressed antigen will usually be detected and resolved from the negative control with most fluorophores. However, an antigen expressed at lower levels may require a fluorophore with a higher intensity such as PE (R-PE) and APC in order to separate positive cells from unstained cells. A guideline for the intensity of various fluorophores is, from brightest to dimmest, PE (R-PE), APC, AF647, FITC, CF-Blue, AF488. 

Another consideration is the colour, and maximum excitation and emission wavelengths of individual fluorophores. Each fluorophore has a particular emission and excitation fluorescence spectra, and therefore differ at the wavelength(s) at which they emit light. This ranges from blue and violet fluorophores such as AF350 and CF-Blue to red dyes including APC and Cy5. As a result, multiple different fluorophore-conjugated antibodies against different proteins of interest can be used to sort cells into several different clusters.

Fluorophore

Colour

Excitation Max (nm)

Emission Max (nm)

AF350

Blue

346

442

AF488

Cyan-green

495

519

AF555

Yellow-green

555

565

AF594

Orange-red

590

617

AF647

Far-red

650

665

APC

Red

650

660

APC-C750

Far-red

650

774

CF-Blue

Blue-violet

405

450

Cy3

Yellow-Green

550

570

Cy5

Red

650

670

DyLight 405

Violet

400

420

DyLight 488

Green

493

518

DyLight 549

Yellow

562

576

DyLight 594

Orange

593

618

DyLight 634

Red

638

658

DyLight 649

Red

654

673

DyLight 680

Far-red

692

712

DyLight 800

Near-infrared

777

794

FITC (Fluorescin)

Green

495

519

PerCP

Blue-cyan

447

678

PE (Phycoerythrin)

Orange-red

565

578

TRITC

Orange

557

576

Texas Red

Red

596

615


Flow Cytometry Products

Abbexa offers a wide range of antibodies and cell detection kits for flow cytometry analysis using the fluorescence detection method. This includes fluorophore-conjugated primary and secondary antibodies, and our apoptosis detection kits and reagents for the differentiation of normal cells and apoptotic cells using cell membrane-based and mitochondrial-based assays. In these apoptosis assays, Annexin V fluorescent conjugates (e.g. Annexin V- FITC) distinguishes apoptotic cells from normal cells. More information about our recommended protocols for flow cytometry can be found here and about our apoptosis kits here.