introduction to flow cytometry by noha kamel. flow cytometry is a method of measuring multiple...

Introduction To Flow Cytometry

ByNoha Kamel

Flow cytometry is a method of measuring multiple physical and chemical characteristics of particles by optical means.

What is flow cytometry?

• Peripheral blood, Bone marrow cells• Bacteria• Yeast

Flow Cytometry is a technology that Flow Cytometry is a technology that simultaneously measures and then analyzes simultaneously measures and then analyzes multiple physical characteristics of single multiple physical characteristics of single particles, usually cells, as they flow in a fluid particles, usually cells, as they flow in a fluid stream through a beam of light.stream through a beam of light.

It measures and analyzes particles (cells) It measures and analyzes particles (cells) according to:according to:– Relative sizeRelative size– Relative granularity or internal complexity Relative granularity or internal complexity – Relative fluorescence intensityRelative fluorescence intensity

• Flow cytometry integrates electronics, fluidics, computer, optics, software, and laser technologies in a single platform.

Fluorescence Activation Process (or Immunofluorescence)

FITC FITC

FITC

FITC

FITC

FITC

Antibodies recognize specific molecules in the surface of some cells

But not others

When the cells are analyzed by flow cytometry the cells expressing the marker for which the antibody is specific will manifest fluorescence. Cells who lack the marker will not manifest fluorescence

Antibodies are artificially conjugated to fluorochromes

Antibodies

Cellular Parameters Measured by Flow

• No reagents or probes required (Structural)– Cell size(Forward Light Scatter)

– Cytoplasmic grabularity(90 degree Light Scatter)

• Reagents are required.– Structural

• DNA content• DNA base ratios• RNA content

– Functional• Surface and intracellular

receptors.• DNA synthesis• DNA degradation (apoptosis)• Cytoplasmic Ca++• Gene expression

Intrinsic Extrinsic

• Cell size.

• Cytoplasmic granularity.

• Cell surface antigens (phenotyping).

• Apoptosis.

• Intracellular cytokine production.

• Intracellular signalling.

• Gene reporter.

• Cell cycle, DNA content, composition, synthesis.

• Bound and free calcium.

• Cell proliferation

• Cell sorting, single cell cloning

Applications of Flow Cytometry.

A suspension of single cells or other particlesin a suitable buffer, usually PBS.

Typical density : 105 - 107 cells / ml

+

Incubate Acquire

Phenotyping, Size and granularity detection:

Sample requirements:

LaserLight488nm

FL2 PE

SSC

FSC

Fluidics

Electronics

Computer

FL3 PerCP, Cy5

FL1 FITC

A cytometer consists of:

Light detectors

Size and Granularity

Size

Gra

nu

lari

ty

Light Scattering, 2 Parameter Histogram

Forward Light Scatter (FLS)

90 degree Light Scatter

Bigger

More Granular

Live Cells

Bigger Cells

Dead Cells

Apoptotic Cells

X Axis

Y Axis

Presenting and Interpreting Data

Dual parameter data can be displayed in two dimensions using dot, density or contour plots.

Single parameters can be displayed as a histogram.

1 Parameter Histogram

1 2 3 4 6 7 150 160 170 .. 190

Channel Number

Positive

Negative

BrighterDimmerCount

1

4

6

Fluorescence picked up from the FITC PMT

2 Parameter Histogram

FITC FL

PE FL

Negative Population

Single Positive FITC Population

Single Positive PE Population

Double Positive Population

Gating and Statistics

• Data generated in flow cytometry is displayed using Multiparamater Acquisition and Display software

platforms.• Histograms corresponding to each of the parameters of interest

can be analyzed using statistical tools to calculate percentage of cells manifesting specific fluorescence, and fluorescence intensity.

• This information can be used to look at fluorescence expression within subpopulations of cells in a sample (gating).

Flow Cytometry Data

Smaller Region, Live cells mostly

Larger Region includes all cells

Gating:

Flow cytometry data analysis. Left-hand plot show a one-colour histogram plot of CD8 expression by peripheral blood lymphocytes. Approximately 38% of events fall between the marker boundaries, and are therefore regarded as CD8 +ve. The centre plot also shows CD8 expression on PB lymphocytes, but depicts the relationship between CD8 and the T-cell marker CD3. The right-hand plot shows a population of CD34 +ve 'stem cells' plotted against side scatter.

• Cellquest• WinList

Available analysis software

Flow Cytometry : Reference Material

Books• Practical flow Cytometry Howard M Shapiro. • Flow Cytometry: A Practical Approach MG Ormerod.• Introduction to Flow Cytometry, JV Watson.• Flow Cytometry: First Principals, Alice Givan.• Cytometric Analysis of Cell Phenotype and Function, McCarthy & Macey.

Journal• Cytometry : Journal of the International Society for Analytical Cytology. www.cytometry.org

• ISAC (International Society For Analytical Cytology) www.isac-net.org

• Salk Institute http://flowcyt.salk.edu

• Purdue University www.cyto.purdue.edu

• Scripps Research Institute http://facs.scripps.edu/index.html

• Cancer Research UK http://science.cancerresearchuk.org/sci/facs

Flow Cytometry on the web