Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Extracellular matrix wikipedia , lookup
Tissue engineering wikipedia , lookup
Cytokinesis wikipedia , lookup
Cell growth wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cellular differentiation wikipedia , lookup
Cell culture wikipedia , lookup
Cytoplasmic streaming wikipedia , lookup
Introduction to Flow Cytometry Shonna Johnston Centre for Inflammation Research University of Edinburgh What is Flow Cytometry • It is the technique used to measure properties of individual cells as they flow in a stream one by one past a sensing point • This ability to measure cellular parameters based on light scatter and fluorescence and to physically purify subpopulations has led to widespread use of this instrumentation in biology and medicine Main Components of Cytometer • Fluidics • Optics • Electronics Fluidics LASER SHEATH FILTER FLOW CELL WASTE SAMPLE Hydrodynamic Focusing FLOW CELL 488nm LASER SAMPLE Fluidics Affect of sample pressure differences Sheath Pressure is constant at 4.5psi LO MED HI 4.6psi 4.8psi 5.0psi Sample Pressure Note: Sample pressure is always higher than Sheath pressure to allow the sample to enter the flow. Optics Forward Scatter / Side Scatter Refracted 488nm light Small cell FSC diode detector 488nm Blue Laser Large cell Blocker bar Prevents the laser light blinding the detector 488/10 BP Allows light between 483-493 IE the refracted laser light This diagram shows how a larger cell size gives a higher forward scatter reading. However remember that this can be affected by the orientation of cells past the laser eg elongated shape of some epithelial cells and any surface variations ie dendrites Optics FACSCalibur FL-1 530/30 BP (515-545) SSC 90/10 Beam Splitter 488/10 BP (483-493) FL-2 DM 560 SP 585/42 BP (564-606) FL-4 DM 640 LP 661/16 BP (653-669) Half Mirror 670 LP FL-3 Fluorescence collection lens 488nm Laser Blue 635nm Laser Red (only used with FL4) Flow Cell FSC 488/10 BP Electronics • The light falling on the photodetector surface generates a current which produces a pulse • This pulse can be processed into height, width and area • Normally the height is displayed on the plots although it is becoming more common to use area •The electronics also allow the adjustment of spectral overlap by compensation equations Electronics 488nm Spectral Overlap FL1 FL2 Compensation Compensation Flow Sorting • Ability to physically separate cells with different properties •Uses a stream-in-air flow which allows droplet formation • Cells are found in droplets which can then be charged • By using oppositely charged plates the drops are pulled to the side into a collection tube Cells • Primary Cells • lymphocytes, neutrophils, eosinophils, monocytes, dendritic cells, platelets • Cell Lines • Transfectants (e.g. CHO, COS7) • Leukaemic cell lines (e.g. MUTU, U937, THP1) • Bacteria • E. coli 0157 Applications • • • • • • • • Phenotyping Apoptosis studies Phagocytosis/Binding Studies Cell Cycle/Kinetics Rare populations - CD34+, side population Intracellular cytokines Intracellular calcium Multiplex cytometric bead assays Links / Further Reading • http://www.cyto.purdue.edu E-mail Forum for cytometry question Lecture presentations • http://flowcyt.salk.edu Links to all major flow lab sites • Practical Flow Cytometry (2004) Howard Shapiro •Flow Cytometry; A Practical Approach (2000) Michael Ormerod