1. Flowcytometry

2. What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell
Metry ~ measure
Measuring properties of cells while in a fluid stream

4. Applications of Flow Cytometry.
Cell size.
Cytoplasmic granularity.
Cell surface antigens (Immunophenotyping).
Apoptosis.
Intracellular cytokine production.
Intracellular signalling.
Gene reporter (GFP).
Cell cycle, DNA content, composition, synthesis.
Bound and free calcium.
Cell proliferation
Cell sorting, single cell cloning

5. Principle of Flow Cytometry
Cells in suspension
Cells flow in single-file
Intercepted by light source(s) (laser)
Scatter light and emit fluorescence
Signal collected, filtered and
Converted to digital values
Storage on a computer
Fluidics
Optics
Electronics
Data display and analysis

6. Basic Principles of Flow Cytometry
Single cell or particle suspension
Fluorescent dyes or Abs that can be attached to an antigen or protein of interest
Flow cell, sheath fluid and a focused laser beam

7. The Flow Cell
The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing.
Sheath
Cell
Sample Stream

8. Basic Principles cont’d
Light is either scattered or absorbed when it strikes a cell
Light scatter is dependent on the internal structure, size and shape.
Forward scatter = size of the cell
Side Scatter = complexity of the cell

9. Forward Scatter
Laser Beam
FSC Detector

10. Side Scatter
Laser Beam
FSC Detector
Collection
Lens
SSC
Detector

11. Side scatter
Forward scatter
Granulocytes
Monocytes
Lymphocytes

12. Why Look at FSC v. SSC
Since FSC ~ size and SSC ~ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogeneous cell population
FSC
SSC
Lymphocytes
Granulocytes
Monocytes
RBCs, Debris,
Dead Cells

13. Side scatter
Forward scatter
Lymphocytes
Monocytes
Granulocytes

14. FLOW CYTOMETRY - Cells are labeled with fluorescent antibodies
directed against cell surface molecules
- Using different color fluorochromes allows
counting of many markers simultaneously
and allows identification of several markers
on the same cell ( Multiparameter Flow)
- In the instrument, cells pass one-by-one past
a laser to excite the fluorochromes and
there are detectors for each type of
fluorochrome

15. - cells are labeled with fluoresence antibodies
Flouresent tag
Surface of a cell, e.g., a lymphocyte
(in solution)

16. What Happens in a Flow Cytometer

17. Fluorochrome

18. Basic Principles cont’d
Fluorescent dyes absorb light of a specific wavelength and reemit light of a different wavelength
Fluorescent signals are detected by PMT and amplified
Optical filters are used to steer light of specific wavelengths to the photo detector
Reflected
Dichroic Filter
Passed
Short or Long Pass Filter
Band Pass Filter
Adsorbed
Absorption Filter

19. Detectors
There are two main types of photo detectors used in flow cytometry
Photodiodes
Used for strong signals, when saturation is a potential problem (eg. FSC detector)
Photomultiplier tubes (PMT)
More sensitive than a Photodiode, a PMT is used for detecting small amounts of fluorescence emitted from fluorochromes.

20. Electronics
Electrical pulses are digitized, the data is stored (‘list mode data’), analysed and displayed through a computer system.
The end result is quantitative information about every cell analysed
Large numbers of cells can be processed quickly

21. THE OPERATOR SETS “GATES” DEFINING
POSITIVE AND NEGATIVE FOR EACH MARKER.
CD19
CD3
CD19
CD3
CD CD19+
CD CD3 +
CD19- CD19-
CD3- CD3+
CD CD3+
Therefore, you can define each cell counted with regard to CD19 or CD3 positivity. Note that there are not normally cells in the circulation that express both T and B cell surface markers.