1. Basic Principles, Instrumentation, and Practices
Flow Cytometry
Basic Principles, Instrumentation, and Practices

2. Introduction/Basic Facts
Laser-optics-computer based technology for measuring the characteristics of biological particles
Bioparticles can be whole cells or prepared cellular constituents
Uses a one particle at a time approach for analysis rather than measuring a bulk property
Measures the light scattering and fluorescence properties of particles

3. Biological Cell
5-20 µm in diameter

4. Cell Labeling Techniques
Antibodies Conjugated to Fluorochromes
FITC, Phycoerythrin, proprietary
Cytoplasmic Dyes/Stains
Permeant, nonpermeant
Nuclear stains
Membrane dyes

5. Most Common Cell Labeling
Single Antibody
Dual Antibody
Internal

6. Flow Cytometry Applications
Detection of Intracellular Cytokine Production
Detection of Intracellular/intranuclear antigens
Estimation of cell viability
Cell transmembrane potential measurements
Measurement of oxidative metabolism
Measurement of environmental particulate uptake
Detection of intracellular cyclins

7. List of Flow Cytometry Applications
Pharmacokinetic monitoring
Quantitation of proteins inserted into membranes
Quantitation of electropermeabilization
Cell cycle analysis
Analysis of apoptosis
Cell Sorting
Chromosome sorting
Up to 7 fluorochrome analysis

8. 1969 Los Almos Labs created the first flow cytometer
Normally have a dedicated operator
USF researchers have access to the Moffitt Core Flow Cytometry Facility

9. Flow Cytometer Block Diagram

10. Spectral Overlap

11. Compensating for Spectral Overlap

12. Control Samples Compensation – for 2 or more colors Negative
Unlabeled cell – zero reference point
Isotype control – nonspecific binding
Positive – make sure that labeled antibody is functional

13. Examples of Flow Cytometric Analysis
One parameter FSC analysis for cell size
One parameter FL1 analysis for drug uptake
Two parameter fluorescence analysis for dual labeled cells
Cell Sorting

14. One Parameter FSC Analysis

15. One Parameter FL1 Analysis

16. One Parameter FL1 Analysis

17. Fluorescence Two Color Analysis
Unfused
Fused

18. Fluorescence Two Color Analysis

19. Cell Sorting
Filters/ detectors
+/- V
+/- V