Flow Cytometry Technical Tips and Calibration Particles

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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
?ow cytometer. Ideally, one cell at a time ?ows
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
?uorescence 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 ?ow cytometry, cell
sorters can physically separate and purify cells
of interest based on their optical
characteristics. Cell Counting
Diagnosis of Health Disorders Such as Blood
Cancers
Determine Cell Characteristics Function
Protein Engineering Detection
Biomarker Detection
Cell Sorting
Detect 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
Fluorchrome Emission Color Excitation Max (nm) Excitation Laser Line (nm) Em-Max (nm)
AF488 Green 495 488 519
FITC (?uorescein) 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 speci?c CD
  (clusters of differentiation) molecule, there are
  usually several monoclonal antibodies with
  different clone numbers. The more often a clone
  is used for ?ow 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
  ?uorescent dyes to antibodies signi?cantly
  exacerbates background binding, especially for
  antibodies labeled by a labeling kit without
  puri?cation. 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 ef?ciently, 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
  ef?ciency. Custom conjugated antibodies with BSA
  and azide-free packaging may be required. When
  using a conjugated antibody, the ratio of
  ?uorochrome to protein (F P, represents the
  degree of labeling) of the ?uorescent 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
  ef?ciency. Custom conjugated antibodies with BSA
  and azide-free packaging may be required. When
  using a conjugated antibody, the ratio of
  ?uorochrome to protein (F P, represents the
  degree of labeling) of the ?uorescent 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 ?xing time to reduce the risk of
  auto?uorescence leading to high background
  ?uorescence. It is recommended to run matched
  unstained cells with the sample to assess
  auto?uorescence.
• It is strongly recommended to use viability dyes
  such as PI, DAPI,7-AAD, Annexin V and pSIVA to
  account for non-speci?c binding. Tissue
  dissociation and digestion often lead to cell
  death and high background ?uorescence, 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 ?ow cytometry, the
  speci?c 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 ?xation 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
  ?xation and permeabilization, and the optimal
  antibody titer has been determined under speci?c
  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
  ?uorescent 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 ?uorescent 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 ?ow
  cytometry. First, particles are not cells and do
  not necessarily scatter light like cells.
  Second, the ?uorescence 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 ?uorescence 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 ?uorescence 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
  coef?cient 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 ?ow path of
  the ?ow 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 ?uorescence. The key is that
  these particles are provided at a de?ned
  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 ?uorescent and non-?uorescent
  forms, but ?uorescent 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 ?uorescent 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 ?ow cytometers. We also have size standard
  particles with diameters range from 3.0-17.9 µm.
  They are a group of uniform and non?uorescent
  polystyrene microspheres, which can provide
  reliable size control, and the cell size can be
  predicted by the forward light scattering (FSC)
  measurements.
• References
• 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 ?ow cytometry. Cold
  Spring Harbor Protocols, 2016(11),
  pdb-prot087288.
• Wang, L., Hoffman, R. A. (2017).
  Standardization, calibration, and control in ?ow
  cytometry. Current protocols in cytometry,
  79(1), 1-3.

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