ROYAL MICROSCOPICAL SOCIETY
FLOW CYTOMETRY COURSE
13-17 September 1999, Sheffield
Supported by: Becton-Dickinson, Coulter, Cytomation, Dako, Genetics Research Instrumentation, Molecular Probes and Phoenix Flow Systems.
ORGANISERS:
DR. MICHAEL G. ORMEROD, Reigate
DR. JOHN LAWRY, Institute for Cancer Studies, Sheffield
Venue: Institute for Cancer Studies, University Medical School, Sheffield
DRAFT PROGRAMME
Module 1. Basics of flow cytometry
Module 2. Clinical Applications
Module 3. Measurement of tumour cell kinetics
Module 4. Applications in cell biology
Module 5. Cell sorting
Module 6. Laser scanning cytometry
BASICS OF FLOW CYTOMETRY
MONDAY 13th SEPTEMBER
09.00 Late registration.
09.15 Welcome to Sheffield. JOHN LAWRY.
09.30 Lecture. INTRODUCTION. MICHAEL ORMEROD (What is flow cytometry? Basic layout of the flow cytometer. Sample preparation. Flow cells).
10.30 Coffee
11.00 Lecture. FLUOROCHROMES AND OPTICAL FILTERS. MICHAEL ORMEROD (What is fluorescence. Energy transfer. Fluorescence quenching. Properties of the more important fluorochromes. Properties of optical filters).
11.45 Lecture. INTRODUCTION TO DNA ANALYSIS. MICHAEL ORMEROD
12.15 Lunch.
13.30 Lecture. ANTIBODY LABELLING AND IMMUNOFLUORESCENCE. MICHAEL ORMEROD
14.30 Practical Demonstration 1. THE FLOW CYTOMETERS (including ROUTINE CHECKS OF PERFORMANCE - Amplifier linearity, sensitivity. JOHN LAWRY & MICHAEL ORMEROD & the manufacturers’ representatives.
15.30 Tea
16.00 Lecture. DETECTORS & SIGNAL PROCESSING. MICHAEL ORMEROD (From the photomultiplier to the computer).
17.45 Lecture. MEASUREMENT OF CYTOPLASMIC AND NUCLEAR ANTIGENS. JOHN LAWRY.
18.30 Close.
TUESDAY 14th
09.00 Lecture. DATA ANALYSIS. MICHAEL ORMEROD
09.45 Practical Demonstration 2 - MULTIPARAMETER LABELLING - FLUORESCENCE COMPENSATION (Peripheral blood lymphocytes - direct conjugates - two and three colours - colour compensation). MARK LOWDELL, Royal Free Hospital Medical School, London, MICHAEL ORMEROD & the manufacturers' representatives.
10.45 Coffee
11.15 Lecture. QUANTIFICATION OF IMMUNOFLUORESCENCE. MARK LOWDELL
11.45 Lecture. INTRODUCTION TO PRACTICAL ON DNA ANALYSIS. MICHAEL ORMEROD
12.15 Lunch
13.30 Practical Demonstration 3. ANALYSIS OF DNA. MICHAEL ORMEROD
15.00 Tea
15.30 Practical Demonstration 4. COMBINED MEASUREMENT OF DNA AND A CYTOPLASMIC ANTIGEN. JOHN LAWRY.
16.30 End of Module
17.00 Special Guest Lecture. MEASUREMENT IN MEDICINE. JAMES V.WATSON, CAMBRIDGE
18.00 End of lecture.
18.45 Sherry reception
19.45 Course Dinner
MODULE 2.
CLINICAL APPLICATIONS09.30 Lecture. IMMUNOPHENOTYPING LEUKAEMIAS AND LYMPHOMAS. RICARDO MORILLA
10.15 Lecture. APPLICATIONS IN BLOOD TRANSFUSION. STEVE GARNER, National Blood Service-East Anglia, Cambridge
11.15 Coffee
11.45 Lecture. MONITORING IMMUNE FUNCTION BY FLOW CYTOMETRY. MARK LOWDELL
12.30 Lunch
13.30 PRACTICAL DEMONSTRATIONS
5A. IMMUNOPHENOTYPING LEUKAEMIAS. RICARDO MORILLA
5B. OTHER CLINICAL APPLICATIONS. TERRY HOY
15.15 Tea
15.45 Lecture. FURTHER CLINICAL APPLICATIONS. TERRY HOY, Cardiff.
16.30 Lecture. CROSSMATCHING AND T CELL MONITORING IN TRANSPLANTATION. BRIAN SHENTON, Newcastle.
17.00 Lecture. QUALITY CONTROL. VIV GRAINGER AND IAN STOREY, Sheffield
18.00 End of module
09.30 Lecture. DNA MEASUREMENT AND CELL CYCLE ANALYSIS. MICHAEL ORMEROD.
10.30 Coffee
11.00 Lecture. CELL CYCLE KINETICS OF TUMOURS USING THE BrdU/ANTI-BrdU METHOD AND ITS CLINICAL APPLICATION. GEORGE WILSON, Gray Laboratory Cancer Research Trust, Mount Vernon, Middx..
12.00 Practical Demonstration 5B. CELL CYCLE KINETICS USING BRDU/ANTI-BRDU LABELLING OF TUMOURS. GEORGE WILSON.
13.00 Lunch.
14.00 Lecture. MEASUREMENT OF ANTIGENS ASSOCIATED WITH PROLIFERATION (cyclins, etc.) JOHN LAWRY.
14.45 Data demonstration. FURTHER MULTI-PARAMETER MEASUREMENTS (inc. RNA, RNA proliferation, measurements on fine needle aspirates, DNA analysis of histological material). MICHAEL ORMEROD.
15.15 Tea
15.45 Practical Demonstration 6B. MEASUREMENT OF ANTIGENS ASSOCIATED WITH PROLIFERATION. (PCNA, cyclins). MICHAEL ORMEROD & JOHN LAWRY.
17.00 Lecture. FLOW ON THE INTERNET. TERRY HOY
18.00 End of module
THURSDAY 16th
APPLICATIONS IN CELL BIOLOGY09.00 Lecture. CELL CYCLE KINETICS USING BrdU/HOECHST-PI LABELLING. MICHAEL ORMEROD
09.45 Lecture. FLOW CYTOMETRY IN MICROBIOLOGY. GERHARD NEBE-VON-CARON, Unilever Research, Colworth, Sharnbrook, Beds.
10.45 Coffee
11.15 Lecture. FURTHER APPLICATIONS TO CELL AND MOLECULAR BIOLOGY. MICHAEL ORMEROD (. Measurement of intracellular pH & calcium. Monitoring electroporation. Monitoring cell fusion. Measurement of membrane potential, drug resistance).
12.00 Lecture. ANALYSIS AND SORTING OF CHROMOSOMES. NIGEL CARTER, Sanger Institute, Cambridge.
13.00 Lunch.
14.00 Lecture. STUDYING APOPTOSIS BY FLOW CYTOMETRY. MICHAEL ORMEROD.
15.00 Tea
15.30 Practical Demonstration 7. APOPTOSIS. MICHAEL ORMEROD, LOUISE TRURAN & JOHN LAWRY
A. SUB-G1 PEAK.
B. ISEL ASSAY FOR DNA STRAND BREAKS.
C. ANNEXIN V LABELLING.
D. DYE UPTAKE.
E. MITOCHONDRIAL MEMBRANE POTENTIAL
17.30 Lecture LASER SCANNING CYTOMETRY. SEAN TYACKE
FRIDAY 17th
CELL SORTING09.00 Lecture. PRINCIPLES OF CELL SORTING. MICHAEL ORMEROD.
09.45 Lecture. ANALYSIS OF RARE EVENTS. TERRY HOY.
10.30 Coffee
11.00 Short presentations. HIGH SPEED SORTERS.
11.45 Practical Demonstration 9. CELL SORTERS IN ACTION. Becton-Dickenson FACSORT and FACS Vantage, Coulter Elite ESP, Cytomation Mo-Flo
13.00 Lunch
14.00 Practical demonstration 9, continued.
15.30 Tea and close.
09.30 Lecture. APPLICATIONS OF LASER SCANNING CYTOMETRY. (TBA)
10.30 Coffee
11.00 Practical demonstration I
12.30 Lunch
13.30 Practical demonstration II
16.30 Close.