Flow Cytometry: Technical Tips and

Calibration Particles

CD

Flow cytometry is a technique used to detect and measure the physical and chemical characteristics
of a group of cells or particles (Figure 1). In this process, a group of cells or particles is suspended in a
liquid and then injected into a flow cytometer. Ideally, one cell at a time flows through the laser beam,
and the light scattered in the laser beam is unique to the cell and its components. Cells are usually
labeled with fluorescence so that the light is absorbed and emitted into a wavelength band. Tens of
thousands of cells can be quickly detected and the data collected is processed by a computer. Flow
cytometry is an instrument that provides quantitative data. Similar to flow cytometry, cell sorters can
physically separate and purify cells of interest based on their optical characteristics.

Cell
Counting

Diagnosis of
Health Disorders
Determine Cell Such as Blood
Characteristics & Cancers
Function

Protein
Biomarker Engineering
Detection Detection

Cell
Detect
Sorting
Microorgan-
isms

Figure 1. Flow cytometry is routinely used in basic research, clinical practice, and clinical trials.

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 Tips for Fluorophore Selection

Emission Excitation Excitation Laser Em-Max

Fluorchrome
Color Max (nm) Line (nm) (nm)

AF488 Green 495 488 519

FITC (fluorescein) Green 493 488 525

AF430 Green 434 405 541

PE (R-Phycoerythrin) Yellow 496. 565 488 575

PE/TR Orange 496. 565 488 613

PI (Propidium lodide) Orange 305, 540 325, 360, 488 620

7-AAD (7-aminoactinomycin D) Red 546 488 647

APC (allophycocyanin) Red 645 595, 633, 635, 647 660

AF647 Red 650 595, 633, 635, 647 668

PE/Cyanine5 Red 496, 565 488 670

PerCP Red 482 488 675

PE/Cyanine5.5 Far Red 496, 565 488 690

PerCP/Cyanine5.5 Far Red 482 488 690

PE/Cyanine7 Infrared 496, 565 488 774

APC/Cyanine7 Infrared 650 595, 633, 635, 647 774

Technical Tips for Choosing Flow Cytometry Antibodies

• Try commonly used clone numbers. For a specific CD (clusters of differentiation) molecule, there are
usually several monoclonal antibodies with different clone numbers. The more often a clone is used for
flow cytometry, the greater the chance that the experiment will be successfully completed.

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• High SI (staining index) ensures good separation of positive and negative cell populations, especially in
experiments that require high resolution.

• Although isotype antibodies or blocking agents are commonly used, suboptimal coupling of fluorescent
dyes to antibodies significantly exacerbates background binding, especially for antibodies labeled by a
labeling kit without purification. Low background binding makes it much easier to identify positive and
negative cell populations.

• Due to their size and inability to cross the plasma membrane efficiently, tandem dyes are recommended
for extracellular staining only.

• For direct labeling of antigens, it is recommended to use conjugated antibodies rather than paired primary
and secondary antibodies. Common buffer additives can interfere with the coupling reaction and limit the
reaction efficiency. Custom conjugated antibodies with BSA and azide-free packaging may be required.
When using a conjugated antibody, the ratio of fluorochrome to protein (F: P, represents the degree of
labeling) of the fluorescent dye and the protein of interest should be calculated.

• For direct labeling of antigens, it is recommended to use conjugated antibodies rather than paired primary
and secondary antibodies. Common buffer additives can interfere with the coupling reaction and limit the
reaction efficiency. Custom conjugated antibodies with BSA and azide-free packaging may be required.
When using a conjugated antibody, the ratio of fluorochrome to protein (F: P, represents the degree of
labeling) of the fluorescent dye and the protein of interest should be calculated.

• Indirect detection is more sensitive and important for effectively identifying low-abundance antigens and
rare epitopes. If no signal is received after using an unconjugated primary antibody, check the species of
the secondary antibody.

• For indirect detection, the cross-species reactivity of secondary antibodies is often a problem. Antibody
labeling kits eliminate the need to use secondary antibodies, resulting in reduced number of incubation
and washing steps while eliminating background caused by cross-species reactions.

Tips for Reducing High Background Fluorescence

• It is best to use fresh cells or cells with a shorter fixing time to reduce the risk of autofluorescence leading
to high background fluorescence. It is recommended to run matched unstained cells with the sample to
assess autofluorescence.

• It is strongly recommended to use viability dyes such as PI, DAPI,7-AAD, Annexin V and pSIVA to account for
non-specific binding. Tissue dissociation and digestion often lead to cell death and high background
fluorescence, so it is important to distinguish between viable and dead cells during analysis (Figure 2).

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 Figure 2. Annexin V/PI staining guidelines.

• Increase buffer capacity, the number of washes, and/or wash times, especially if high background is
observed when using unconjugated primary antibodies. Alternatively, the antibody titer may be too high
and further dilution of the antibody may be required.

• When facing high background staining, using Fc receptor blocking reagents can avoid unwanted bindings
between Fc region of the antibody and the Fc-receptors. Increasing the concentration or exposure time of
such reagents would help too.

• The use of detergents can cause high background staining. For intracellular targets, the use of alcohol
permeabilization is a good alternative.

Tips for No Signal or Weak Fluorescence Intensity

• If the signal is weak, the detection antibodies may be too diluted. Although primary antibodies have been
validated for flow cytometry, the specific cells, tissue types, or experimental conditions may require titration
of antibody concentration.

• If no signal is detected, the target may be not accessible. Check the predicted location of the protein and
whether the fixation and permeabilization methods are correct for the target. To prevent the internalization
of surface antigens, cells should be kept on ice during the assay. In some cases, you can optimize the
staining effect by adjusting the incubation temperature or staining time.

• If there is no problem with protocols of target fixation and permeabilization, and the optimal antibody titer
has been determined under specific experimental conditions, verify if any pretreatment of the cells (such
as stimulating immune cells) is required to induce or enhance the target molecule expression.

• If the targets are secreted proteins, make sure inhibitors such as Brefeldin A or monensin are used. These
compounds prevent the export of newly synthesized proteins by disrupting the ER-Golgi transport
mechanism and eventually capture the proteins in their respective cellular compartments. These inhibitors
are needed when evaluating cytokines.

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• For adherent cells that use trypsin to separate cells from the surface, the cause of the weak signal may be
related to the effect of trypsin on the expression of extracellular molecules. Sodium azide prevents the
modulation and internalization of surface antigens. If cryopreserved cells are used, check if the target
antigen is affected by the freezing and/or thawing process.

• Check the excitation and emission spectra of the fluorescent dyes used. Make sure all lasers are properly
aligned, as misalignment can cause weak signals. The use of calibration beads can help calibrate
instrument performance for each channel.

• Excessive light during the dyeing process results in photobleaching of fluorescent dyes and dissociation of
tandem dyes. Make sure the sample is protected from light as much as possible.

Calibration Particles for Flow Cytometry

There are two important factors to keep in mind when using manufactured particles (i.e. calibration
particles) instead of cells for flow cytometry. First, particles are not cells and do not necessarily scatter light
like cells. Second, the fluorescence of particles may be similar to that of cells stained with a particular dye,
but is almost never exactly the same.

• Aligning Particles
When installing and building the instrument, manufacturers use particles with multiple sizes and/or
fluorescence levels to ensure optical alignment. Moreover, personally track the performance of the
instrument after installation and before preventive maintenance are necessary. When running the
alignment particles, in order to obtain the same mean fluorescence intensity (MFI) reading, one needs to
be aware of any changes in the applied voltage. If the reading deviation appears on all detectors of a
given laser, it may indicate a loss of laser power. In addition, pay attention to the changes in the coefficient
of variation (CV) of the particle population. An increase in CV means a reduction in sensitivity, and there
may be a misalignment of the laser or problems with detection optics.

Creative Diagnostics applies alignment particles that are used to check whether the flow path of the flow
cytometry is aligned and the inside of the instrument is clean or clogged. DiagPoly™ Ultral Multiple
Fluorescent Polystyrene Particles have enhanced UV and Far Red Fluorescence intensity than DiagPoly™
Multiple Fluorescent Polystyrene Particles, and the latter is suitable for alignment of FITC, PE, PE-TR, and
PE-Cy5 channels.

• Counting Particles

Counting particles are beads of various sizes with or without fluorescence. The key is that these particles
are provided at a defined concentration. This allows the setup of a stop gate to get a certain number of
events and use of those events to calculate the concentration of cells in the original sample. These
counting particles come in fluorescent and non-fluorescent forms, but fluorescent beads are often the
best. Fluorescence makes them very easy to gate in comparison to gating only scattered signals.

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Creative Diagnostics offers fluorescent particles of diameters from 0.05-1.9 µm with increased forward light
scatter sensitivity. They are designed to characterize microparticles (0.5-0.9 µm), aquatic bacterial
(0.2-0.6 µm), and platelets (0.9-3 µm), which provides a submicron size standardization tool for flow
cytometers. We also have size standard particles with diameters range from 3.0-17.9 µm. They are a group
of uniform and nonfluorescent polystyrene microspheres, which can provide reliable size control, and the
cell size can be predicted by the forward light scattering (FSC) measurements.

References:
1. Crowley, L. C., Marfell, B. J., Scott, A. P., & Waterhouse, N. J. (2016). Quantitation of apoptosis and necrosis
by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harbor Protocols, 2016(11),
pdb-prot087288.
2. Wang, L., & Hoffman, R. A. (2017). Standardization, calibration, and control in flow cytometry. Current
protocols in cytometry, 79(1), 1-3.

For more information,

view our website: www.cd-bioparticles.com

Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

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