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Flow cytometry and cell sorting

Course content:
The course is designed to equip those having only limited or no prior experience of flow cytometry with a basic grounding in the principles of the technique and working knowledge of its practice with special emphasis on the application of flow cytometry to cell sorting.

Organizer:  Assoc. prof. Soren Hansen, Dept. of Cancer & Inflammation, University of Southern Denmark, Odense 

ECTS credits: 2.4

Number of participants: Max. 8

Teachers: Morten Løbner (ML), BD Bioscience, Denmark; Claus H. Nielsen (CHN), Copenhagen University Hospital, Ulrik S. Jakobsen (USJ), Dept. Clinical Immunology, Odense University Hospital; Inger Andersen (IA); Soren Hansen, University of Southern Denmark

Location: Winsløwparken 19-21, University of Southern Denmark, Odense

Course fee
The course is free of charge for PhD students enrolled in The Institute of Health Sciences at the University of Southern Denmark.
Students enrolled in Universities that have joined the "Open market agreement"  should pay for course material, which in this case amounts to DKK 4399,-
For other participants there is a course fee of DKK 13.104,-

Registration

Example of programme:

Day 1
09.00-09.15 Introduction
09.15-10.45 Flow cytometry 1 The basic principles
                                   - The flow system
                            - Laser excitation                   
                            - Light scatter and fluorescence
                            - Multiple fluorochrome compensation
                            - Autofluorescence
                            - Data analysis
                            - Quality controls

10.45-11.00 Coffee

11.00-12.30 Flow cytometry 2 The basic practicalities
                             - The cell preparation
                             - The parameters for investigation (membrane glycoproteins, surface lipids, intracellular DNA, free Ca2+, oxidative 
                                metabolites etc.)
                             - Labelling with fluorochromes (incl. choosing the right marker-fluorochrome
combination)
                             - Data acquisition and software
                             - Gating
                             - Positive and negative controls

12.30-13.15 Lunch

Practical Sessions Winsløwparken 21, 1st floor, Flow Cyto Lab
13-15-16.15 Exercise 1 The basics
                              - Starting up
                              - Instrument settings
                              - Calibration with standard beads
                              - Compensation
                              - Maintenance

Exercise 2 Identification of cell sub-populations in a blood leukocyte preparation 
                                      - Morphological discrimination
                              - Identification with the help of surface markers (CD3,       CD19 etc.)

Day2.(Theoretical sessions)
09.00-10.00 Flow cytometry 3 Antibodies as probes
                               - The antibody-antigen interaction
                               - Monoclonal vs. polyclonal antibodies
                               - Direct vs. indirect labelling
                               - The biotin-streptavidin connection
                               - Extracellular vs. intracellular probing (fixation and permeabilisation)

10.00-10.15 Coffee

10.15-11.15 Flow cytometry 4 Other probes 
                                       - DNA staining
                               - Free calcium staining
                               - Staining for membrane depolarization
                               - Staining for oxidative metabolites
                               - Staining for detection of cell division
                               - Staining for markers of apoptosis


11.15-12.15 Flow cytometry 5 Quantitative flow cytometry
                                       - Quantitation cell sub-populations
                               - Quantitation of surface markers
                               - Quantitation of ligand-receptor interactions
                               - Cell-cycle analysis

12.15-13.00 Lunch

13-15-16.15 Practical Sessions Winsløwparken 21, 1 st floor, Flow Cyto Lab
                 Exercise 3 Viability assessment 
                               - Apoptosis vs. necrosis (PI + Annexin V)
                 Exercise 4 Assessment of cell proliferation
                               - CFSE approach

Day 3
(Theoretical sessions)

09.00-11.45 *Flow cytometry 6 Cell sorting
                                - Basic considerations
                                - Important Factors for Successful Cell Sorting
                                           Quality of the cell preparation
                                           Cell density
                                           Sorting rate v. viability
                                           Purity versus yield
                                           “Invisible” impurities
                                           Sorting strategies
                                - Alternatives to phenotype sorting - side population sorting, reporter genes                   
                                - Practicalities: The time factor
                                - Recovery – the harsh reality                   
                  * including 15 min. coffee break
11.45-12-15 Discussion of results - Exercises 3 and 4

12.15-13-00 Lunch

Practical Sessions Winsløwparken 21, 1 st floor, Flow Cyto Lab
13.00-16.00 Exercise 4 (part Demonstration) Introduction to the cell sorter                          
                 Exercise 5 (part Demonstration) A rare event sort



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