Bi9393e Analytical cytometry
Institute of Biophysics, CAS
Kralopolska 135
612 65 Brno
E-mail: ksoucek@ibp.cz
tel.: 541 517 166
Karel Souček, Ph.D.
Lukáš Kubala, Ph.D., doc.
Hana Sedláček Polášková, Ph.D.
Cytometry
◼ Cytometry is the collective name for a group of methods used to measure various
characteristics of cells. Variables that can be measured by cytometric methods include
cell size, cell number, cell morphology (shape and structure), cell cycle phases, DNA
content, and the presence or absence of specific proteins on the cell surface or in the
cytoplasm. Cytometry is used to characterize and count blood cells in routine blood
tests such as a complete blood count. Similarly, cytometry is also used in cell biology
research and in medical diagnostics (for example, to detect cancer or AIDS).
There are different types of cytometry:
◼ Flow cytometry
◼ Spectral flow cytometry
◼ Hyperspectral cytometry
◼ Image cytometry
◼ Mass cytometry
◼ In vivo cytometry (non-invasive cytometry)
Ref. What is Cytometry?. International Society for Advancement of Cytometry. 2013-03-28
Two common ways to quantify the total
number and type of cells in a sample
Microscopy
Provides details of cell morphology for tens or
hundreds of cells. Can provide information about
cellular interactions.
Shape
Distribution of
components in the cell
Flow Cytometry
Quantifies a high number parameters of cells in suspension
for hundreds or thousands of cells per second and is capable
of sorting living cells.
Size
Surface and intracellular components
Granularity
BD technology and innovation
a, Representative flow cytometry plots (left) and
percentage of degranulating NK cells isolated from the
blood of ten healthy donors (right), as measured by
surface CD107a, in response to uninfected and ZIKVinfected
JEG-3 cells (8 h coculture, E:T ratio 1:3). b,
NK cell-specific killing of uninfected and ZIKV-infected
JEG-3 cells. c, ER stress, as assessed by XBP1 splicing
(left) and increases in BIP (middle) and CHOP (right)
mRNA, in JEG-3 cells that were uninfected or infected
with ZIKV, HSV-2 or human cytomegalovirus (HCMV) for
1–2 days or treated with tunicamycin (Tu) for 1 day.
Indicated samples were pretreated with the ER stress
inhibitor salubrinal (n = 3 samples). mRNA levels, as
assayed by quantitative PCR with reverse transcription
(RT–qPCR), were normalized to ACTB. d, Effect of
salubrinal pretreatment of target cells on NK cell killing
of ZIKV-infected (top) and tunicamycin-treated (bottom)
JEG-3 cells (n = 6 samples). e, Effect of NKR-blocking
antibodies (Ab) on NK cell killing of uninfected or ZIKVinfected
JEG-3 cells (n = 3–7 samples). Ctrl, control. f,
Specific killing of the classical NK cell target
722.221 cells, or of uninfected or ZIKV-infected JEG-
3 cells by human NK cell line YT cells knocked out
for NCR1 or NCR3 or treated with control single-guide
RNAs (n = 3–6 samples). g, Representative flow
cytometry histogram (left) and mean fluorescence
intensity (MFI) of NKR–Ig fusion protein (NKp46–Ig and
NKG2D–Ig) binding to uninfected or ZIKV-infected JEG-
3 cells (right) (n = 3 samples). b,d–f, Specific killing
assessed by 8 h 51Cr release assay using an E:T ratio of
10:1 unless otherwise indicated. Data are mean ± s.e.m.
of at least three independent experiments or technical
replicates. Statistics were performed using two-tailed,
nonparametric, unpaired t-test (a,b), one-way analysis
of variance (ANOVA) (c), two-way ANOVA (e–g) or area
under the curve followed by one-way ANOVA (d).
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
-CD56 is a single transmembrane glycoprotein also known
as N-CAM (Neural Cell Adhesion Molecule), Leu-19, or
NKH1. It is a member of the Ig superfamily. The 140 kD
isoform is expressed on NK cells and NK-T cells. CD56 is
also expressed in the brain (cerebellum and cortex) and at
neuromuscular junctions.
-lysosomal-associated membrane protein-1 (LAMP-1 or
CD107a) has been described as a marker of CD8+ T-cell
degranulation following stimulation.
Flow cytometry
For surface staining, cells were stained for 30 min on ice in the dark
with LIVE/DEAD-Violet stain (1:1,000) and then with primary
antibodies for 15–30 min in PBS and 2% FCS (followed by secondary
antibodies, when applicable, for 20 min). For protein–Ig staining, cells
were incubated with 50 µg ml–1 fusion protein for 1 h at 4 °C and then
stained with fluorescent-anti-human IgG for 1 h. Cells were fixed in 1%
paraformaldehyde (Affymetrix) for 10 min before flow cytometry. Flow
cytometry was assessed on gated live cells (Supplementary Fig. 1). For
intracellular staining, cells were fixed and permeabilized using the
CytoFix/CytoPerm kit. One of the treated samples was used for isotype
staining, and MFI of staining with the isotype control antibody was
subtracted from MFI of the specific antibody. Analysis was performed
on a FACSCanto II (BD). BD FACSDiva 8.0 (BD) software was used for
data collection, with analysis performed using FlowJo v.10.4.2
(TreeStar).
Course structure
◼ Lectures
– 8 lectures on flow cytometry and applications
– 2 lectures on microscopy techniques and on the basics of image analysis
– 2 lectures of student presentations
◼ Bi9393ec Analytical Cytometry- practical labs, 3 days in a block
– Follows lectures on flow cytometry, in block groups
◼ Test
– The course will conclude with an exam in the form of a test summarizing the material
covered throughout the semester. The result of the test will constitute 75% of the
overall grade.
– Seminar
• Each student will present a short seminar whose topic will be consulted
with the lecturer and will be related to the focus of the course. Credit
will be awarded on the basis of this presentation, and the seminar‘s
grade will also be reflected in 25% of the overall grade.
Seminar of students
◼ Seminar topic: How I use/want to use/could
use analytical cytometry methods in my
DP/DSP.
◼ The aim is to demonstrate an understanding
of the underlying principles and their
application in biology.
◼ The presentation must be prepared e.g. in
PowerPoint. It is recommended to submit the
presentation in advance to the lecturer for
consultation and review.
◼ The length of the presentation is 5 minutes
(~2 min introduction of the essence of your
experimental work) + discussion
Information resources - flow
cytometry
◼ Practical Flow Cytometry, Howard M. Shapiro, Wiley-Liss; 4th edition
◼ Cytometry: New Developments, Volume 75, Fourth Edition (Methods in
Cell Biology), Zbigniew Darzynkiewicz, Academic Press; 4th edition
◼ Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and
Genomes; Jaroslav Dolezel (Editor), Johann Greilhuber (Editor), Jan Suda
(Editor), February 2007
◼ Single Cell Analysis, J. Paul Robinson, Andrea Cossarizza, 2017
For more information on flow cytometry books, please visit:
http://www.cyto.purdue.edu/flowcyt/newbook.htm
https://www.beckman.com/resources/reading-
material/ebooks/practical-flow-cytometry
Howard Shapiro
https://archive.org/details/PracticalFlowCyt
ometryShapiro
Information resources - flow
cytometry (methods and protocols)
◼ The Handbook of Flow Cytometry Methods
◼ Current Protocols in Cytometry
◼ Company web pages, e.g.
– https://www.thermofisher.com/cz/en/home/references/protocols.html
– https://www.thermofisher.com/cz/en/home/references/protocols/cell-and-
tissue-analysis/flow-cytometry-protocol.html
– https://www.cellsignal.com/learn-and-
support/protocols?_requestid=3668092
Information sources - cytometry
(journals)
◼ Cytometry Part A
https://onlinelibrary.wiley.com/journal/15524930
◼ Cytometry Part B: Clinical Cytometry
https://onlinelibrary.wiley.com/journal/15524957
Information resources - microscopy
◼ Taatjes D. J. Cell Imaging Techniques, Methods and Protocols,
Humana Press, Totowa, New Jersey, 2005
◼ Stephens D. Cell Imaging, Scion Publishing Ltd., 2006.
◼ Pawley, J. (Ed.), Handbook of Biological Confocal Microscopy, 3rd ed.,
2006
◼ Fluorescence Microscopy: From Principles to Biological Applications
◼ Ulrich Kubitscheck (Editor), ISBN: 978-3-527-32922-9, 2013, Wiley-
Blackwell
Information resources - (Internet)
◼ Purdue University, Cytometry Labs
http://www.cyto.purdue.edu/
◼ International Society for Advancement of
Cytometry
http://www.isac-net.org/
◼ Excyte
https://expertcytometry.com
◼ https://x.com/ISAC_CYTO
◼ https://x.com/flowcytometryUK
◼ https://x.com/FlowJoNow
◼ https://x.com/CSAC_CZ
◼ https://www.csac.cz
General introduction to flow
cytometry
◼ Basic principles, possibilities of flow
cytometry and its application
◼ History
These particles have something in
common...
Algae
ChromosomesBlood cells
Protozoa
... certain parameters of these particles can be
measured by flow cytometry. bd_logo
Commercial equipment and development
What is a flow cytometer?
What can we analyse with flow
cytometry?
◼ Count particles in suspension
◼ Quantify light scattering, and
fluorescence intensity at the single
cell level
◼ Evaluate 10*5 to 10*6 particles in less
than 1 minute
◼ Physically separate individual particles
(defined populations and single cells) for
further analysis
Adapted from J.P. Robinson Purdue University BMS 631
What are the principles?
◼ Light scattering (Light scatter) using a laser or
UV lamp
◼ Specific fluorescence or full spectrum
detection
◼ Hydrodynamically focused particle stream
◼ Electrostatic separation of particles
◼ Multivariate data analysis capability
Adapted from J.P. Robinson Purdue University BMS 631
Definition of
◼ Flow cytometry
– Measuring the properties of flowing
particles (cells)
– also known as Fluorescence-Activated
Cell Sorting (FACS)
◼ Flow sorting
– physical separation of particles (cells)
based on parameters measured by flow
cytometry
Technical components
◼Light sources
◼Detection systems
◼Fluid system
◼Separation
◼Data collection
◼Data analysis
J.P. Robinson Purdue University BMS 631
Technical components
◼Detection systems
Photomultiplier Tubes (PMTs)
previously 1-2
now > 8
Diodes previously light scatters now also detect fluorescence
◼Light sources
Lasers (350-363, 420, 457, 488, 514, 532, 600, 633 nm)
Argon ion, Krypton ion, HeNe, HeCd, Yag
UV (Arc) Lamps
Mercury, Mercury-Xenon
J.P. Robinson Purdue University BMS 631
The non-technical part is equally
important...
◼ (in)specific brands/sondas
◼ Antibodies
◼ biomarkers
◼ preparation, processing of
material/samples/tissues
◼ ...
History of staining of biological
materials
Until the mid-19th century - only natural dyes were used
Anton van Leeuwenhoek used saffron to stain muscle cells in
1719
www.euronet.nl/users/warnar/leeuwenhoek.html
History of staining of biological
materials
Paul Ehrlich - 1879 used acidic and basic dyes to
distinguish acidophilic, eosinophilic and neutrophilic
leukocytes
Clin Lab Med. 1993 Dec;13(4):759-71.
The Ehrlich-Chenzinsky-Plehn-
Malachowski-Romanowsky-Nocht-
Jenner-May-Grunwald-Leishman-
Reuter-Wright-Giemsa-Lillie-Roe-Wilcox
stain. The mystery unfolds.
Woronzoff-Dashkoff KP.
The Principle of the Fluorescence Microscope - August Köhler -
ix70biglightpaths
http://micro.magnet.fsu.edu/primer/anatomy/anatomy.html
History of staining of biological
materials
Friedman
Friedman (1950) - combined acid fuchsin,
acridine yellow and berberine for the
detection of uterine tumour cells using a
fluorescence microscope
Acid Fuchsin
Acid magenta
Acid ruby
Acid roseine
Absorption Max 540-545
von Bertalanffy & Bickis
Ludwig von Bertalanffy (1901-1972)
von Bertalanffy & Bickis (1956)
- Acridine orange metachromatic fluorescence was used to detect RNA in tissue
- they also used it to distinguish between normal and tumor cells
Absorption Max 467 nm
J.P. Robinson Purdue University BMS 631
Immunodetection
History:
1940 - Conns, immunofluorescent labeling of cryosections
1959 - Singer, development of the method of conjugating antibodies with a marker
1966 - Graham&Karnovsky, the enzyme labelling method (HRP)
1974 - Taylor& Burns - routine immunohistochemistry
1975 - Kohler& Milstein - production of monoclonal antibodies using hybridoma
Stain Your Own Cell
P.J. Crossland-Taylor
"Sheath Flow" principle
"Provided there is no turbulence,
the wide column of particles will
then be accelerated to form a
narrow column surrounded by
fluid of the same refractive index
which in turn is enclosed in a
tube which will not interfere with
observation of its axial content."
J.P. Robinson Purdue University BMS 631
Wallace Coulter
◼ Wallace Coulter Coulter
orifice - 1956 ◼
(patent 1953) measurement
of
conductivity change
during passage of cells in
suspension through a
small hole
Original patent
application of W.
Coulter 1953
Photo by J.Paul Robinson
J.P. Robinson Purdue University BMS 631
How to count cells?
◼ Hemocytometer (Bürker chamber) was the standard
for cell counting until ~1950
◼ Dimensions are 3x3x0.1 mm. Usually red blood cells
(1 x 106 /mm3 ) are counted after dilution 1:200
◼ Leukocytes (5x103 /mm3 ) are diluted 1:10 in red blood
cell lysing solution
◼ Statistical error:
– coefficient of variance (CV) is 4.4% with 500 cells counted
– pipetting and dilution error is ~10%
Adapted from J.P. Robinson Purdue University BMS 631
Roche Innovatis
Cedex
CellDrop - Denovix
https://www.denovix.com/celldrop/
Coulter Counter
First commercial version of
CC
Coulter Counter
© CC
Beckman Coulter
◼ Multisizer 3&4 COULTER COUNTER®
Roche Innovatis
CASY TT
Cytograph. A benchtop instrument capable of measuring the light scattering of a
HeNe laser (1970).
Mack Fulwyler- sorter
Mack Fulwyler - sorter 1965 - Los Alamos National Labs - his sorter separated particles
based on electronically measured volume (same principle as Coulter counter) and
separated by electrostatic deflection.
The aim was to sort the red blood cells, because a bimodal distribution of cell volume
was measured. The principle of separation was based on the inkjet printer principle of
Richard Sweet of Stanford (1965).
After it was clarified that red cell bimodality is an artifact, this group was able to separate neutrophils and lymphocytes from blood.
J.P. Robinson Purdue University BMS 631
Richard Sweet
Richard Sweet developed an
electrostatic inkjet printer whose
principle was used by Mack Fulwyler for
his cell sorter.
J.P. Robinson Purdue University BMS 631
Mack Fulwyler- sorter
Mack Fulwyler- sorter
K. Soucek
Leonard Arthur "Len" Herzenberg
Key "cytometric" publications
◼ 1934: Moldovan - Photoelectric measurement of cells in a capillary
◼ 1947: Gucker - photoelectric cell counting
◼ 1949: Coulter's particle computer
◼ 1961: Rotman uses fluorescence for the first time to quantify an enzymatic reaction
◼ 1964: Sweet - electrostatic inkjet printer
◼ 1965: Fulwyler - May 1965 - patent for electrostatic sorter
◼ 1965: Kanetsky - spectrophotometric measurement of cells
◼ 1965: Fulwyler - November 1965 - publication on cell separation in Science magazine
◼ 1968: Gohde - first article on fluorescence flow cytometry (in German)
◼ 1969: Gohde - patent
◼ 1969: Van Dilla - second paper on fluorescence flow cytometry
◼ 1969: Mullaney - first paper on the description of light scattering as a cytometric parameter
◼ 1969: Heryenberg - third paper on fluorescence flow cytometry
◼ 1973: Gohde - double marking patent
◼ 1977: Gohde - description of signal compensation in double marking
◼ 1978: Kachel - flow imaging - combination of flow cytometry and image analysis
◼ 1983: isolation and detection of nuclei (DNA) from paraffin-embedded tissues
◼ 1984: congress on DNA cytometry nomenclature
◼ 1987: Graz - high-speed chromosome sorting
◼ 1991: Robinson - automation of clinical systems - flow cytometer and barcode reader
Source - ISAC 2006 - The Wall of History
What's it all for... like...
What's it all for... like...
◼ 38 million people worldwide are infected with HIV (WHO, 2019)
◼ ~0.7 million people die annually from HIV/AIDS, 1.7 million newly infected (there are ~11 million AIDS orphans in Africa)
◼ CD4 T cell quantification was/is a key parameter in disease/treatment monitoring, since ~1985
◼ Since 2016, WHO recommended that all people living with HIV be provided with lifelong ART, including children,
adolescents and adults, and pregnant and breastfeeding women, regardless of clinical status or CD4 cell count. By the
end of 2019, 185 countries had already adopted this recommendation, covering 99% of all people living with HIV globally.
◼ Flow cytometry is the "gold standard"
◼ Optimized procedures and equipment for low-cost (<3 EUR/sample) and fast detection (250 samples/day)
◼ Aydogan Ozcan: "Kill the cost, safe life"
Flow Cytometry On-a-Chip
◼ MAGNETIC COUNTING
– RESEARCHER: Hakho Lee, Assistant Professor of Radiology, Harvard Medical School
PROJECT: Enumerating and characterizing circulating tumor cells
PROBLEM: Circulating tumor cells (CTCs) are incredibly rare, with just a handful per milliliter of human blood.
SOLUTION: Lee's group fabricated a hybrid microfluidic device out of polydimethylsiloxane (PDMS) that can count CTCs in
real time using tiny sensors called micro-Hall detectors. (Sci Transl Med, 4:141ra92, 2012)
◼ PCR-ACTIVATED SORTING
– RESEARCHER: Adam Abate, Assistant Professor of Bioengineering and Therapeutic Sciences, University of California,
San Francisco
PROJECT: Analysis of rare, uncultivable microbes
PROBLEM: Developing specific antibodies for bacteria that cannot be cultured
SOLUTION: As a postdoc in the Harvard University lab of droplet-based microfluidics pioneer David Weitz, Abate
codeveloped a device that could sort droplets according to their fluorescence intensity. Unlike traditional microfluidics, in
which molecules and cells flow naked through channels, droplet-based devices encapsulate molecules or cells in uniform,
picoliter-scale aqueous reaction chambers encased in oil.
◼ SORTING BY CELL DEFORMABILITY
– PROJECT: Cancer cell phenotyping
PROBLEM: Not every cell type has a known antigen that defines it. Plus, antibody binding may activate receptors,
potentially changing the cell's behaviour.
SOLUTION: A physicist by training, Guck used laser beams in his graduate work to study the physical properties of cells.
Not all cells are equally squishy, he found: while normal cells are relatively rigid, cancer cells are more pliable. "The more
aggressive the cell, the softer it is, and that may be necessary for it to pass into tissues," Guck explains.
◼ RAMAN-ACTIVATED CELL SORTING
– RESEARCHER: Jian Xu, Professor and Director, and Bo Ma, Group Lead of Microfluidics, Single-Cell Center, Qingdao
Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
PROJECT: Microbial biofuels development
PROBLEM: Biofuels R&D requires identifying cells capable of specific carbon chemistries. But as these cells have yet to be
cultured and studied, researchers have few if any molecular hooks for identifying and sorting them.
SOLUTION: The team turned to a label-free method of single-cell interrogation known as Raman-activated cell sorting
(RACS) (Anal Chem, 87:2282-89, 2015).
http://www.the-scientist.com/?articles.view/articleNo/43034/title/Flow-Cytometry-On-a-Chip/
Cellular Indexing of Transcriptomes and
Epitopes by Sequencing (CITE-seq)
What preceded it...and what we
are part of now...
◼ Development of techniques for rapid and
reproducible detection of cytometric
parameters.
◼ New scientific knowledge leading to the
definition of appropriate diagnostic markers.
http://www.cyto.purdue.edu/cdroms/gh/HTML/start.htm?loc=http://www.cyto.purdue.edu/cdroms/gh/HTML/video/video.html?v=Flowtheinvention.wmv
http://www.cyto.purdue.edu/cdroms/gh/HTML/start.htm?loc=http://www.cyto
.purdue.edu/cdroms/gh/HTML/video/video.html?v=Flowtheinvention.wmv
ISAC presents: Mack Fulwyler Innovator,
Inventor & Pioneer
http://www.cyto.purdue.edu/cdroms/cyto10a/seminalcontributions/fulwyler.html
http://www.cyto.purdue.edu/cdroms/cyto10a/seminalcontributions/fulwyl
er.html
https://www.youtube.com/watch?v=3s5l2mepKkY
https://www.youtube.com/yt/brand/media/image/YouTube-logo-full_color.png
State-of-the-Art
Two common ways to quantify the total
number and type of cells in a sample
Microscopy
Provides details of cell morphology for tens or
hundreds of cells. Can provide information about
cellular interactions.
Shape
Distribution of
components in the cell
Flow Cytometry
Quantifies a high number parameters of cells in suspension
for hundreds or thousands of cells per second and is capable
of sorting living cells.
Size
Surface and intracellular components
Granularity
BD technology and innovation
BD CellView Image Technology, provides the features
of both and more
High speed, high-parameter flow
cytometry
High-content images providing cell morphology and
cellular interactions
Rapid analysis and sorting of cell populations defined
by traditional flow parameters combined with new
image parameters
BD TECHNOLOGY AND INNOVATION
January 21st, 2022
BD TECHNOLOGY AND INNOVATION
64
Real-time cell images
65
Lysed whole
blood
Mouse over any data point and
see the cell image pop-up
HeLa cells
Image data and all flow data are
merged and correlate one to one
Real time display of gated
events on image wall
BD TECHNOLOGY AND INNOVATION
Summary of the lecture
◼ Introduction to the course
◼ Sources of literature
◼ History of flow cytometry
◼ Basic principles
At the end of today's lecture, you should:
1. know what the requirements are for this course
2. know the basic sources of information
3. have a brief overview of the history of flow cytometry
4. be familiar with some basic principles of flow cytometry
At the end of today's lecture, you should:
1. know what the requirements are for this course
2. know the basic sources of information
3. have a brief overview of the history of flow cytometry
4. be familiar with some basic principles of flow cytometry