MACS Handbook

Flow cytometry basics

1 Introduction

Flow cytometry is the measurement of chemical and physical properties of cells as they “flow” one by one
through an integration point, most commonly a laser. As cells scatter laser light in different directions
(forward or to the side), intrinsic cellular properties, such as relative cell size and cytoplasmic complexity, can
be measured. In human whole blood, for example, lymphocytes, monocytes, and granulocytes can be
distinguished simply because they scatter laser light differently.

Light scatter profile of lysed whole blood
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Typical light scatter profile of lysed human whole blood.

Most modern flow cytometers can also measure the expression of cell surface or intracellular markers,  nucleic acid content, enzyme activity, and much more. To probe these cellular properties, fluorescent reagents are used, such as fluorochrome-conjugated antibodies. These reagents have characteristic light emission properties so that they can be detected separately in distinct fluorescence channels.
A unique feature of flow cytometry is that fluorescence on a cellular or particle level can be measured very

rapidly. As cells or particles travel through the light path, the fluorescent probes are excited, resulting in the
detection of the emitted light and ultimately of the specific cellular properties at a rate of 10,000 events/second.

Excitation and emission spectra
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Excitation and emission spectra for VioBlue, FITC, and APC.

Flow cytometry is used in both clinical and basic research. Many research fields benefit from this
technology, including immunology, transplantation, hematology, neuroscience, stem cell research, cancer
research, cell biology, molecular biology, drug discovery, systems biology, marine biology, microbiology,
virology, and bioprocessing. Clinical applications are, among others, in vitro diagnostics, clinical trials, or

monitoring patients after treatment. Some clinics also use flow cytometry to characterize donor and recipient
cells prior to transplant to lower the risk of adverse effects following transplantation.

Examples of flow cytometry applications 

  • 7-color immunophenotyping
  • Cell viability staining and apoptosis detection with PI and annexin-V conjugates
  • Intracellular staining for cytokines and transcription factors
  • MACS® Cytokine Secretion Assays
  • Cell cycle analysis using PI and or BrdU
  • Microvesicle analysis
  • Cell enumeration
  • Measurement of calcium fluxes using Fluo-4
  • Detection of oxygen-reactive (H2DCFDA) and NO-reactive (DAF-FM diacetate) species
  • Cell proliferation analysis with CFSE or CMFDA
  • Fluorescent reporter protein detection – GFP, CYP, YFP, tdTomato, mCherry, RFP
  • Analysis of virus particles, bacteria, mitochondria

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2 Components of a flow cytometer

To enable the light scatter and fluorescence measurements of cells and particles, MACSQuant® Instruments
utilize a combined system of fluidics, optics, and electronics.

  • Fluidics: a liquid flow system to align and move cells or particles through a laser path.
  • Optics: a series of optical filters and mirrors to direct light of specific wavelengths emitted by cells or
    particles towards detectors, which amplify the light signal.
  • Electronics: interface between optics and computer, converting the amplified light signals
    proportionally into voltage, thus enabling graphical plotting.

2.1 The fluidic system

Hydrodynamic focusing is used to align and move the cells or particles through the laser path. The sheath
fluid flows through the flow cell with constant pressure. The cells are injected into the middle of the
sheath fluid flow. By principles of laminar flow, the cell sample and sheath fluid flow in the same direction but
stay separate. The pressure differential between the sheath and the cell sample determines how wide the
sample stream is as it flows through the flow cell. The lasers excite the cells or particles individually and the
scatter and fluorescence light are measured.

Illustration of hydrodynamic focusing of the MACSQuant® Instrument.

MACSQuant Instruments utilize a 0.5 mL pump syringe for volumetric uptake and subsequent sample
injection into the flow cell. The syringe pump–driven fluidics draws the specific sample volume into the uptake needle. The uptake needle then aligns with the flow cell injection port (unless running an enrichment
protocol) and injects the specific volume at the programmed fluidic speed.
For flow applications that require very tight population resolution and CV of the measured peaks, e.g., cell
cycle analysis, low fluidic speed is recommended, as the sample stream is the narrowest. For general
immunophenotyping applications, any fluidic speed is appropriate. It is important, however, that the event rate setting of the instrument does not exceed 10,000 events per second for any of the fluidic speeds.

2.2 The optical system

Filters and mirrors

After excitation by a laser, fluorescent reagents bound to cells or particles emit light, which is detected
according to their specific wavelength range. When using a combination of fluorescent dyes, these reagents should have distinct emission spectra. MACSQuant Instruments utilize various optical filters and dichroic mirrors to direct light of specific wavelengths to the according fluorescence detectors. This arrangement creates “fluorescence channels”.
There are different types of filters that are used to define which wavelengths can enter the fluorescence
channels. According to their optical properties they are designated long-pass, short-pass, and band-pass filters. MACSQuant Instruments use long-pass and band-pass filters.

Optical filters
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Optical filters
Illustration of light of different wavelengths passing through long-pass (LP), short-pass (SP), and band-pass (BP) filters.

Long-pass filters are denoted by the letters LP and a specific number, e.g., LP 750. This means that light with a wavelength of 750 nanometers (nm) and longer can pass through the filter. Light of all other wavelengths will be absorbed. Short-pass filters have a similar designation, e.g., SP 500. In that case, light of wavelengths of 500 nm and shorter can pass through, while the remaining light will be absorbed. Band-pass filters allow light of a specific wavelength range to pass through. Band-pass filters are referred to as BP followed by two numbers. The first number represents the midpoint of the wavelength range. The second number specifies the wavelength range in nm that can pass. Thus, light that can pass through a BP 525/50 filter ranges from 500 to 550 nm.
Dichroic mirrors have essentially the same function as filters, but are oriented at a 45° angle to the light path, whereas filters are oriented perpendicular to the light path. The light that is not able to pass through a mirror is deflected at a 90° angle towards another light detector. 

With this optical setup, eight distinct fluorescence channels and two scatter channels are created by combinations of the light emitted after excitation by the three lasers, as well as various filters and mirrors.

Photon detectors – Photomultiplier tubes (PMTs)
The light paths of specific wavelengths, i.e., fluorescence channels, are monitored by so-called photomultiplier tubes (PMTs). PMTs amplify the fluorescence signal emitted from the fluorochrome bound to the cell or particle.
The magnitude of amplification is dependent on the voltage applied to each PMT. As the voltage is increased, the amplification of the detectable fluorescence is increased, resulting in an increased mean fluorescence intensity (MFI). Conversely, as the voltage is decreased, the amplification is decreased, resulting in a lower MFI.
As each channel’s PMT detects and amplifies the light emission of a specific fluorescent reagent, it is
recommended to assign the correct nomenclature to these channels. The PMT not only amplifies the
detectable light, but converts the optical signal into a voltage pulse that is relayed to the electronics for signal
conversion and display.

Analog-to-digital conversion
Analog signals are converted into digital signals for display of data according to the bit depth of the analog-to-digital converter (ADC) of the flow cytometer. The MACSQuant Instruments have a 16 bit ADC for all parameters. However, for the pulse area, an additional calculation allows for 18-bit processing. This means that for the pulse area measurements , the signals are resolved into over 253,000 bins. This allows for a five-decade logarithmic scale for data display.

2.3 The electronics

The electronics of a flow cytometer convert the light signals into proportional electronic signals (voltage
pulses), digitizes the signal according to the ADC bit depth, and interfaces with the computer for data transfer.

MACSQuant Instruments are digital flow cytometers, which means that the conversion from analog to
digital signals happens at the point of signal detection and amplification. Advantages of digital flow
cytometers include:

  • Voltage pulse height, width and area can all be a measureable parameter
  • Compensation of spectral overlap is applied after digital conversion of signal, meaning compensation can
    be re-adjusted any time post acquisition.
  • Detection thresholds or triggers can be set on any of the detection channels.
  • Fluorescence values detected as zero or negatives will be measured as such and can be displayed in the
    plot choices.

Voltage pulses
As a cell is passing through the laser path, the attached fluorochrome will begin to fluoresce. This light is
detected by the PMT and starts the generation of a voltage pulse. As the cell fully enters the laser path, the
fluorescence reaches peak emission, resulting in the highest peak of the voltage pulse. Finally, as the cell
leaves the laser path, this detectable fluorescence decreases, finishing the voltage pulse.

Voltage pulses
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Voltage pulses
A voltage pulse can be characterized by its height (signal intensity), width (time of flight), and area (emitted fluorescence while the cell passes through the laser path).

3 Visualization of flow cytometry data

3.1 Visualization of flow cytometry data

For data visualization, values of each measured parameter can be plotted in various ways. These plots can
be univariate (histograms) or bivariate (dot plots).

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3.1.1 Univariate plots – Histograms
Univariate plots allow the visualization of one optical parameter versus event number. The measured values
for each event are stored in a data file, comprised of a table of stored signal intensities (of fluorescence and
scatter signals) for each measured parameter. As events start to accumulate, a “curve” is generated that
describes the distribution of the measured events according to their signal brightness. Looking at the scale
from left to right, the intensity of the plotted signals increase. Populations on the left side of the scale have
lower signal intensity than the populations on the right side of the scale.
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A univariate histogram plot describes distribution of intensity.

3.1.2 Bivariate plots – Dot plots
Dot plots allow the display of two optical parameters in one graph. The position of an event is determined by
two values, one for the x- and one for the y-axis. When all events are plotted, events with similar intensity
values accumulate into clouds or populations.
Dot plot
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Dot plot
Bivariate dot plots allow visualization of two optical parameters simultaneously.

3.1.3 Bivariate plots – Density plots
Density plots display two parameters as a frequency distribution similar to a dot plot (each cell is represented
as a dot). Additionally, they depict the distribution of cells within a population with a very high density of
events. In the figure below, the red color represents the highest density within a population. With decreasing
density, the color transitions from yellow over green to blue.
Density plot
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Density plot
A density plot depicts the distribution of cells within a population.