The document presents an overview of fluorescence-activated cell sorting (FACS), a technique for isolating individual cells using fluorescent dyes and a flow cytometer. It details the components and principles of FACS, including cell labeling, sorting methods based on size and fluorescence intensity, and applications in cancer and immunology research. The document also provides tips on sample preparation to ensure optimal sorting outcomes.

1. Fluorescence-activated Cell Sorting
FACS
Advanced medical instrument
Super.by Dr. Nazeh Al-Abd
Prepared by waleed al-shalabi
Sep.2024

2. Topics covered in this presentation
INTRODUCTION
PARTS OF FLOWCYTOMETER MACHAINE
PRINCIPLE OF CELL SORTING
STEPS FOR CELL SORTING
DATA ANALYSIS
How the cell sorting
collection of sample
Application of FACS
collection of sample

3. Introduction
((
Facs
))
is a cutting-edge technique used to isolate
individual cells from a sample and analyse their
properties
.
It uses fluorescent dyes and flow-cytometer to sort cells based
on various parameter ,including size, surface makers and
fluorescence intensity
Facs allowing researchers to isolate single cells with an
unprecedented accuracy of 99.99%

4. Where are samples
Analyser
run
? Analyser
Analyser
Analyser
Sorter
Analyser

5. A sorter can be divided into 3 main subsystems
:
Fluidics
To introduce and focus the cells for interrogation
for sorting
and create a stable breakoff
Optics
To generate and collect the light signals
Electronics
To convert the optical signals to proportional digital signals, process the signals
and communicate with the computer

6. Fluidics
How can we record individual cells
?
•
The sample is introduced into the running sheath
fluid in the flow cell
In a pressurized fluidics system, cells are
hydrodynamically focused so that they pass
individually through one or more laser beams
Sheath fluid and sample do not mix
Reduction of cross-section causes an accelaration
of the central core stream that leads to laminar
fluidics
Sample flow rate adjustable
•
•
•
•

7. Optics
There are two different optics’ systems in a sorter
:
Excitation optics consisting of
:
•
•
Lasers
Fibre optic cables that carry beams to steering prisms, which then direct
laser beams to the stream

8. Optics
There are two different optics’ systems in a sorter
:
Collection optics consisting of
:
• Fibre optic cables that direct the emitted light to the appropriate emission
filter block
Filters that direct the signals in the emission block to the appropriate
photon multiplier tube (PMT)
•

9. Optics
Filters are arranged in different arrangements

10. Electronics
Photon multiplier tube (PMT)

12. Principle of cell sorting
FACS is based on the principle that cells can be labeled with fluorescent
dyes and sorted according to their fluorescence intensity
FACS is carried out using a flow cytometer, which is a machine that can
measure the fluorescence of cells as they pass through a laser beam
FACS offers many benefits over traditional cell separation techniques,
such as centrifugation and filtration
FACS is much faster than these methods and can often be completed in
minutes
FACS can be used to purify cells that are difficult to separate using other
methods
Finally, FACS can be used to sort cells into multiple groups, allowing for a
more detailed analysis of cell populations

13. The first step in FACS sorting is to label the cells with fluorescent dyes
.
The fluorescent dyes are usually attached to antibodies that bind to
specific cell surface markers. Cells are routinely labelled with PE or FITC
conjugated antibodies. FITC is a green fluorescent dye that is excited by
blue light and PE is a red fluorescent dye that is excited by green light.
The cells are incubated with the fluorescent dyes for 30-60 minutes
After incubation, the cells are washed and diluted using a buffer. The cell
sorting buffer contains salts, proteins, and other molecules that help to
keep the cells healthy during the sorting process. The cells are then
resuspended in phosphate buffered saline (PBS) to remove unbound dye
.
Steps of cell sorting

14. Steps of cell sorting

15. The next step is to pass the labeled cells through a flow cytometer.
The flow cytometer uses lasers to excited the fluorescent dyes and
detectors to measure the fluorescence intensity of the dyes. Based
on the fluorescence intensity, the flow cytometer sorts the cells
into different groups
Steps of cell sorting

16. The cells are sorted into groups according to their fluorescence intensity and
size. The most common sorting method is called forward scatter (FSC) and
side scatter (SSC). The FSC channel sorts cells based on their size, with larger
cells being sorted into the high FSC channel and smaller cells being sorted
into the low FSC channel. The SSC channel sorts cells based on their
granularity, with more granular cells being sorted into the high SSC channel
and less granular cells being sorted into the low SSC channel
Steps of cell sorting
After sorting, the cell groups are collected in tubes or plates for further
analysis. The cell purity can be checked using a microscope or by flow
cytometry. Purity can be calculated by dividing the number of cells in the
desired group by the total number of cells sorted
.

17. Steps of cell sorting

18. Analysis of data

19. Analysis of data

20. Analysis of data

21. Cell sorting

22. How the cell sorting
At a pressure of 70 psi, the sheath and sample fluid are
accelerated to around 35 m/ s very rapidly
.
Cell aggregates must be eliminated from a sort as they
are a potential source of reduced purity
.
cells of interest need to be separated from other cells in the sample.
Drop-based sorters rely on the production of dispersed drops, the
aim being that each drop should contain a single cell or no cell at all
This is done by passing a column of liquid through a small
orifice (nozzle) where the drops are created by vibrating a
piezoelectric device attached to the body of the flow cell. This
applies a standing wave of vibration to the column of fluid

24. Cell sorting
• „
Live“ image of the
stream taken from
the video monitor
.
• Processed image
from the video
capture card
.
• Drops are visible
because the stream
is illuminated by a
stroboscope lamp
• Notice the
ligament extending
from the last
connected drop
and the minor
„
fast“ satellite
drops which will
„
melt“ with the
main drops on
their way down
.
which is driven
the drop drive
frequency
.
by

25. Cell sorting
This is a bad
example: Slow
satellite drops will
never merge and
always give bad side
streams
.
•
It is of no use trying
to sort with this kind
of stream
!
•
Remember to check
the satellite drops.
There should be only
few of them, and
they should be of the
„
fast“ type
.
•

26. Cell sorting
Core stream undeflected

27. Core stream and deflected side streams
Cell sorting

28. Cell sorting
At the very moment when the
cell is analysed, it is still quite
far away from the droplet
formation point
.
Thus, somehow the
information to charge a drop
must be delayed until the cell
of interest has reached this
„breakoff
“
point
.
Cell of interest analysed
Drop delay
The stream has to be
charged when the cell of
interest arrives at the last
droplet. This „delay“ in
charging the stream is called
the drop delay
.
Sort pulse for this cell

29. Cell sorting
Determining the correctand robust drop delay is one of the most important steps
in the machine setup
With a wrong drop delay, no cells or wrong cells reach your collection tube

30. Into what collection devices can I sort
my samples
?
collection of sample

31. Cell sorting - Bulk
Bulk sorts are done with the purpose of
enrichment for cell culture / DNA + RNA Analysis
/
adoptive transfer / purification of cells
fluorescent protein, etc
…
expressing a
Cells can be sorted into
:
• 1.5
ml / tubes (4 populations in parallel)
• 5
ml FACS tubes (4 populations in parallel)
• 15
ml tubes (2 populations in parallel)
• Beaker (1 population)
• Samples can be cooled / uncooled
Collection medium is adjusted to the
experiment (bring it along)

32. Cell sorting - Plates
Plate sorts are done with the objective of
sorting directly into multiwell plates for the
purpose
clonal
of tissue culturing
/ T
ce
ll
and B cell
expansion
/ single DNA/RNA
analysis/ etc
…
Cells can be sorted into
:
• 6
to 384-well plates
Only one population can be sorted

33. Cell sorting - Slides
Slide sorts are done with the purpose of
sorting directly into slides that can be used for in
vitro assays (e.g. Seahorse assay) or microscopy
Cells can be sorted into
:
• Microscopy slides
• Ibidi-type chamber slides
Only one population can be sorted

34. Application of FACS
Cancer Research FACS can be used to isolate cancer cells from a mixed population of
cells
Immunology Research
FACS can be used to study the immune system. It can be used to isolate specific
types of immune cells, such as T-cells and B-cells, from a mixed population
Stem cells Research
This is important because it allows researchers to study the properties of stem
cells and develop new treatments for diseases
.
Developmental biology researche This is important because it allows for the
isolation of specific embryonic cell types that are involved in a particular
stage of development
In research, FACS is often used to isolate rare cell types such as tumor-infiltrating
lymphocytes from cancer patients. In the clinic, FACS is used to diagnose and treat
blood disorders, such as leukemia and lymphoma

35. Tips on sample preparation
Quick tips on sample preparation

36. Tips on sample preparation
• Plan your experiment well and choose your fluorescent antibodies in
agreement with the optical configuration of the intended sorter
.
different sorters may slightly vary in the optical configuration
Note that
• Bring along the following controls
:
 Unstained/non-transfected cells to estimate background signal
 Bring single stained samples (cells and/or beads) to set compensation
 Bring FMOs if needed to set gates

37. Tips on sample preparation
• A maximum of 2% FCS / BSA is recommended in the suspension medium of
cells to be sorted because otherwise cells become sticky
• Avoid phenol-red media due to autofluorescence; most cell types will do well
in just PBS+FCS/BSA
• If your cells have a tendency to for aggregates consider adding one of the
following
:
•
•
use calcium/magnesium free sorting buffer or add EDTA (< 5 mM)
if the cell preparation induces increased cell lysis use 25 µg/ml
DNAse I + 5 mM MgCl2 (no EDTA!)
use 1% Accutase in sorting buffer
•

38. Cell sorting
All samples need to be
filtered, preferably directly
before sorting

39. Cell sorting
For a bulk sort provide cells with following concentrations
used nozzle
:
depending on the
x 106
 70
µm nozzle ⇒ 40 cells /
ml
x 106
 85
µm nozzle ⇒ 20 cells /
ml
x 106
 100
µm nozzle ⇒ 10 cells /
ml
5
x 106 cells / ml
For a single cell sort provide cells with a concentration
The nozzle size should be 3 to 4x bigger than your sorted cells

40. Cell sorting
• The minimum sample volume for sorting is 200 uL
• Samples can be loaded from 1.5 mL Eppendorfs, 5 mL FACS tubes and 15
mL Falcon tubes
• Prepare collection tubes and add sufficient collection medium with regard
to the expected yield / target cell number. You can calculate with following
drop volumes/sort event
:
70
µm nozzle
85
µm nozzle
100
µm nozzle
130
µm nozzle
⇒
⇒
⇒
⇒
~
1
nl
~
1.5
nl
~
2.5
nl
~
8
nl
• The choice of collection medium depends on the downstream
applications and is therefore variable
Volatile harmful substances such as 2-Mercaptoethanol or Trizol are not allowed
[
use instead lysis buffer from a kit (e.g.Rneasy) or sort into RNAlater buffer
]

41. Useful links
:
FCFacility website
:
http://www.cytometry.uzh.ch/en/index-fcf.html
Cell sorting sample preparation guidelines
:
http://www.cytometry.uzh.ch/en/index-fcf/sorters/Sample-prep.html
Info on operator-based sorts
:
http://www.cytometry.uzh.ch/en/index-fcf/sorters/Operator-based-
service.html
Info on power-user training
:
http://www.cytometry.uzh.ch/en/index-fcf/teaching/Sorter-Training.html
BD Biosciences Tools
:
http://www.bdbiosciences.com/us/s/tools
Invitrogen SpectraViewer
:
https://www.thermofisher.com/ch/en/hom
e/life-science/cell-analysis/labeling-

42. THANK YOU