Flow Cytometry: Technical Tips and Calibration Particles
                     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.
Tel: 1-631-633-6938 Email: [email protected]
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 common ly  used clone numbers. For a  specific CD (clusters of  di f ferentiat ion) 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.
Tel: 1-631-633-6938 Email: [email protected]
•High SI ( s ta in ing index)  ensures g o od  separation of  positive a n d  negat ive cell populations, especially in  
experiments that require high resolution.
•Although isotype antibodies or blocking agents are common ly  used, subopt imal  coupl ing 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 t ime 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 r ec o m m en d e d  to use viability dyes such as PI, DAPI,7-AAD, Annexin V a nd  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).
Tel: 1-631-633-6938 Email: [email protected]
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 detect ion antibodies m a y  be  too diluted. Although pr imary 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  m a y  be  not  accessible. Check the predic ted location of  the protein an d   
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 prob lem with protocols of  target  fixation a n d  permeabilization, a nd  the op t ima l  ant ibody 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 Tneele: d1e-d6 3w1h-e6n3 e3v-6al9u3a8ting cytokines. Email: [email protected]
• 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.
Tel: 1-631-633-6938 Email: [email protected]
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-biopart icles.com
Fax: 1-631-938-8221 Address: 45-1 Ramsey Road, Shirley, NY 11967, 
USA 5