Introduction To Flow Cytometry By Noha 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 simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. Flow Cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. It measures and analyzes particles (cells) according to: It measures and analyzes particles (cells) according to: –Relative size –Relative granularity or internal complexity –Relative fluorescence intensity

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

Fluorescence Activation Process (or Immunofluorescence) 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 IntrinsicExtrinsic

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 particles in a suitable buffer, usually PBS. Typical density : cells / ml + Incubate Acquire Phenotyping, Size and granularity detection: Sample requirements:

Laser Light 488nm FL2 PE SSC FSC Fluidics Electronics Computer FL3 PerCP, Cy5 FL1 FITC A cytometer consists of: Light detectors

Size and Granularity Size Granularity

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 Channel Number Positive Negative Brighter DimmerCount 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.

ISAC (International Society For Analytical Cytology) Salk Institute Purdue University Scripps Research Institute Cancer Research UK Flow Cytometry on the web