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1. Objectives
Learning Objectives
At the end of this chapter the students should
be able to:
 Describe the principle of Flow cytometry
 Discuss the difference between intrinsic
and extrinsic parameters in flow cytometry
 Explain the difference between forward
light scatter (FSC) and side scatter (SSC)
 Describe the application of Flow Cytometry

2. Flow Cytometry
Debre Berhan
May 22, 2022

3. Outline
• Introduction to flow cytometry
• Isolation of Lymphocyte Populations

4. What is Flow Cytometry?
• Is an automated system and it Measure properties of
cells in flow
• Multi-parametric analysis is made on individual
particles that are suspended in a fluid
• single cells in a fluid suspension are analyzed in
terms of their intrinsic light-scattering characteristics
and are simultaneously evaluated for extrinsic
properties using fluorescent labeled antibodies
• By using several different fluorochromes,
cytometers can simultaneously measure multiple
cellular properties.

10. Fluorochromes commonly used in flow cytometry
Excitation λ Fluorochrome Emission λ
488nm(argon
laser)
Fluorescein
isothyocyanate
(FITC)
530
Phycoerythrin (PE) 580
Propidium iodide
(PI)
620
Peridinin
chlorophyll(PerCP)
670
633nm(He-Ne
laser )
Allophycocyanin
(APC)
670

11. Example
• Stain cells with:
– FL1: CD4-FITC
– FL2: CD19-PE
– FL3: 7-AAD (live/dead cells)
– FL4: CD3-Alexa Fluor 647
– FL5: CD8-Alexa Fluor 750

17. Data Analysis
17
FL4: CD3-Alexa 750
FL1:
CD4-FITC
FL5:
CD8-Alexa
750
FL4: CD3-Alexa 750
CD4
+ T
cells
CD8
+ T
cells
Cd4+
Cd3-
Cd4-
Cd3-
Cd4-
Cd3+
Cd8+
Cd3-
Cd8-
Cd3- Cd8—
Cd3+