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Understanding Flow Cytometry Basics

Flow cytometry allows for simultaneous measurements of multiple characteristics of individual cells at high speeds. Cells can be analyzed based on their size, granularity, and fluorescence to identify cell types and detect specific molecules through markers. A flow cytometer uses lasers and optical detectors to measure forward scatter, side scatter, and fluorescence of cells labeled with fluorescent antibodies as they flow in a fluid stream. This allows identification of cell populations, quantification of cell types, and sorting of cells. Flow cytometry has many clinical applications such as immunophenotyping and monitoring diseases like leukemia.

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Grace Yuni Soesanti Mh
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60 views28 pages

Understanding Flow Cytometry Basics

Flow cytometry allows for simultaneous measurements of multiple characteristics of individual cells at high speeds. Cells can be analyzed based on their size, granularity, and fluorescence to identify cell types and detect specific molecules through markers. A flow cytometer uses lasers and optical detectors to measure forward scatter, side scatter, and fluorescence of cells labeled with fluorescent antibodies as they flow in a fluid stream. This allows identification of cell populations, quantification of cell types, and sorting of cells. Flow cytometry has many clinical applications such as immunophenotyping and monitoring diseases like leukemia.

Uploaded by

Grace Yuni Soesanti Mh
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd

Principle of Flowcytometry

What Is Flow Cytometry?

Cyto = cell Metry = measurement

 Simultaneous measurements of multiple


characteristics of a single cell
 Measurements are made on a per-cell
basis at routine rates of 500 to 4000
cells per second
What Do All These Things Have
in Common?
algae chromosomes

blood protozoa
cells
Why Do We Analyze Cells?
 Count cells of a particular type
 Count cells which have a particular function
 Determine whether cells are carrying out
their function
 Use monoclonal antibodies to detect specific
molecules
Flow Cytometry – Samples
 Whole Blood
 Bone Marrow
 Tissue / Nodes
 Body Fluids
 Tumor Cells
 Bacteria, Plankton, Viruses
 Plant
Components of Flow Cytometer
 Fluidics
 Laser
 Optics - Flow Cell, Fluorescence Pickup Lens
System, Optical Filters
 Detection - Photomultiplier Tubes (PMTs),
Electronics
 Data Collection, Interpretation and Display -
Computer
Flowcytometry
What Can a Flow Cytometer
Tell Us About a Cell?

 Its relative size (Forward Scatter—FSC)


 Its relative granularity or internal
complexity (Side Scatter—SSC)
 Its relative fluorescence intensity
Properties of FSC
Right Angle Light
and SSC
Detector
a Cell Complexity

Inciden Forward Light


t Light Detector
Source a Cell Surface
Area

Forward Scatter—diffracted light


 Related to cell surface area
 Detected along axis of incident light in the forward direction
Side Scatter—reflected and refracted light
 Related to cell granularity and complexity
 Detected at 90° to the laser beam
Forward Scatter – Size of Cell
Forward Scatter Detector

Voltage Pulse
Side Scatter - Granularity of Cell

LASER

Side Scatter Detector at 90

LASER
lymphocytes
monocytes

neutrophils
Lysed Whole Blood largest and most
complex population

1000
800 Neutrophils
Side Scatter

600
400

Monocytes
200

smallest and least Lymphocytes


complex population
0

0 200 400 600 800 1000

Forward Light Scatter


What is Fluorescent Light?
HO O
l = 488 nm l = 530 nm
C
Incident CO2H Emitted
Light Energy Fluorescent Light
Fluorescein Antibody Energy
Molecule

 The fluorochrome absorbs energy from the laser

 The fluorochrome releases the absorbed energy by:


– Vibration and heat dissipation
– Emission of photons of a longer wavelength
Emitted Fluorescence Intensity
Fluorescence
 Binding Sites
FITC FITC
FITC

FITC
FITC
FITC FITC
FITC
Number of Events

FITC
FITC

Fluorescent Intensity
Collection Optics–
FACSCalibur Cytometer
Detector Filter Color Fluorochrome

FL1 530/30 nm Green FITC

FL2 585/42 nm Yellow/Orange PE

FL3 670nm LP Dark Red PerCP, PerCP-Cy5.5

FL4 661/16 nm Red APC


T B
CD45 Gating
 most informative marker
 good for all cell types
Side Scatter

gran

mono

NRBC lymph
Forward Light Scatter
Characteristics Can Be Measured
Using a Flow Cytometer

Orange
Green

SSC
Time

FSC FSC Green


Time
Data
Processor SSC Orange
Time

Red
Time

Time
Gating
Dual Parameter Histograms
1 2
-/+ +/+
20% 40%
60%
PE
10% 30%
40%
3 -/- +/- 4
FITC

30% 70%
Two-Color Cell Analysis

10 4
upper left = FITC– and PE+ upper right = FITC+ and PE+

10 3
(double positive)
PE
10 2
10 1

lower left = FITC– and lower right = FITC+ and PE–


PE–
0
10

(double negative)
10 0 10 1 10 2 10 3 10 4
FITC
Two-Color Cell Analysis
Cell Sorting

488 nm laser
-+ FALS Sensor
- + +- Fluorescence detector
+
-+ +-
- +-
+ +

Charged Plates - + +
-
-
- +
+ -

Single cells sorted


into test tubes
Waste
Clinical Aplication
 CD4 / CD8 Enumeration
 Lymphocytes subset
 Leukemia Phenotyping
 CD34 Enumeration
 DNA content
 Cell cycle Analysis
THANK YOU

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