PhD course Flow Cytometry & Cell Sorting

This year the flow cyto course was held in connection with 6th International Stem Cell School in Regenerative Medicine 14-19 June, 2009.

(Courses are run by Danish Stem Cell Research Doctoral School (DASCDOC))

General objectives: Objective: This 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. Graham Leslie, Immunology and Microbiology, University of Southern Denmark, Odense.
Duration: 3 days; 17-19 June 2009
ECTS points: 2
Number of participants: Max. 8
Teachers: Henrik Holst (HH), BD Bioscience, Denmark; Claus H. Nielsen (CHN), Copenhagen University Hospital, Ulrik S. Jakobsen (USJ), Dept. Clinical Immunology, Odense University Hospital; Graham Leslie (GL), and Inger Andersen (IA), University of Southern Denmark
Location: Winsløwparken 19-21, University of Southern Denmark, Odense
Application deadline: 1 May 2009
Course fee: estimated 8,500 DKK exclusive of travel and stay, material costs approx. DKK 2,000
Registration: Please fill out course application form (www.dascdoc.dk - Activities, courses) and send it to the DASCDOC secretariat.

Program:

Day 1
09.00-09.15 Introduction GL
09.15-10.45 Flow cytometry 1 The basic principles (USJ)
                    - The flow system
                    - Laser excitation
                    - Light scatter and fluorescence
                    - Multiple fluorochrome compensation
                    - Autofluorescence
                    - Data analysis
                    - Quality control
10.45-11.00 Coffee
11.00-12.30 Flow cytometry 2 The basic practicalities – (GL)
                    - The cell preparation
                    - The parameters for investigation (membrane glycoproteins, surface lipids,
                      intracellular proteins, DNA, free Ca2+, oxidative metabolites etc.)
                    - Labelling with fluorochromes (incl. choosing the right marker-fluorochrome
                      combination)
                    - Data acquisition – the 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 (USJ)
                   - Starting up
                   - Instrument settings
                   - Calibration with standard beads
                   - Compensation
                   - Maintenance
                   Exercise 2 Identification of cell sub-populations in a blood leukocyte preparation
                   (USJ/GL)
                   - 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 (GL)
                   - 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 – (CHN)
                   -  DNA staining
                   - Free calcium staining
                   - Staining for membrane depolarisation
                   - 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 - (CHN)
                   - Quantitation cell sub-populations
                   - Quantitation of surface markers
                   - Quantitation of ligand-receptor interactions
                   -  Cell-cycle analysis
12.15-13.00 Lunch

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

Day 3 (Theoretical sessions)
09.00-11.45 *Flow cytometry 6 Cell sorting (HH)
                   - 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 pehnotype 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 (HH)
                    Exercise 5 (part Demonstration) A rare event sort (HH)