University of Stellenbosch Flow Cytometry Unit

• Part I (Basic Flow Cytometry) offers the beginner a sound basis of flow cytometry within the clinical environment.
• Part II (Clinically Applied Flow Cytometry) and
• Part III (Research Applications in Flow Cytometry)

are intended for the more advanced user.

The cost of each Course is R1000 and includes all reagents, use of equipment, materials, notes, etc.

To maximise the learning experience, candidates are advised to read the details of the topics covered in the three Courses (Part I, Part II, Part III) and follow the links for further information about the hardware, software, and techniques used.

Please contact Mandi Alblas (021) 9389 394 or John Michie (021) 9389 539 to make a booking.

N.B. Inexperienced persons wanting to register for Parts II and/or III must first complete Part I.

2006 Schedule

Part I (details here)

• 20 - 24 February
• 22 - 26 May
• 21 - 25 August

Part II (details here)

• 19 – 23 June
• 4 – 8 September

Part III (details here)

• 3rd - 7th July

Please contact Mandi Alblas (021) 9389 662 or John Michie (021) 9389 539 to make a booking.

Course Details

Part I - Basic Flow Cytometry

MONDAY

• Introduction to Flow Cytometry.
• Safety in the Flow Cytometry Laboratory
• Fluorochromes, Antibody Labeling, Immunofluorescence, Spectral Overlap and Compensation.
• BD FACSCalibur™ Flow Cytometer Start-up and Shut-down Procedures, Maintenance, Fluorochromes/Probes, Acquisition Setup and Compensation. BD FACSComp™ Software and QC
• Practical - BD FACSComp™ Software, Instrument Setup, Calibration and Manual Compensation.

TUESDAY

• Immunobiology - CD4 Monitoring, Absolute Counts
• BD FACS MultiSET™ Software – Manual- and Auto-Gating Principles, Sample Data Acquisition and Analysis.
• Data Management.
• Practical CD4 Sample Preparation using BD MultiTEST™
• Data Acquisition and Analysis with BD FACS MultiSET™ Software
• HIV and CD4 Monitoring

WEDNESDAY

• Revision – Flow Cytometer Calibration and Compensation.
• BD CellQuest™ Pro Software - Setting Up Templates, Sample Data Acquisition and Analysis.
• Cell Surface and Intracellular Staining.
• Practical: Single-Colour and Multi-Colour Labeling of Cell Surface Markers - Analysis using CellQuest™ Pro Software.

THURSDAY

• Practical: Prepare Blood Sample in BD TruCOUNT™ Tubes using BD MultiTEST™ Antibodies.
• Calibrate FACSCalibur and Aquire Sample using BD FACS MultiSET™ and BD CellQuest™ Pro software.
• Calculate Absolute CD4 Count Manually from BD CellQuest™ Pro Data and Automatically with BD FACS MultiSET™.

FRIDAY

• Comparison of BD FACS MultiSET™ and BD CellQuest™ Pro Data, Gating Strategies, QC, Setting Up Reference Ranges, Pitfalls/Trouble Shooting.
• Operational Troubleshooting and Maintenance.
• Review and Discussion of the Course.

Part II Clinically Applied Flow Cytometry

MONDAY

• BD CellQuest™ Pro Advanced, Setting Up Acquisition and Analysis Templates, Designing Gating Protocols, Statistical Analysis and Presentation of Data
• Sample Automation and BD LoaderManager™ Software, Setting Up Protocols using BD Worklist Manager™ Software.
• Practical – BD Worklist Manager™; Instrument Setup, Working with Panels and File Management.

TUESDAY

• CD4 Monitoring, Absolute Counts using BD FACS MultiSET™ and BD CellQuest™ Pro Software, QC, Setting Up Reference Ranges, Pitfalls/Trouble Shooting and Case Studies.
• Non-HIV-Related Immunodeficiencies.
• Antigen/Fluorescence Quantification – CD38 (BD QuantiBRITE™)

WEDNESDAY

• Lymphoma/Leukemia Immunophenotyping, Immunophenotypic Panels (BD Oncomark™), Case Studies
• Combining Surface and Intracellular Detection of Antigens Including Cytokines, Compensation, Sample Data Acquisition, Compensation and Analysis (BD FastImmune™).
• BD PAINT-A-GATE^PRO™ and BD Attractors™ Software for Identification of Populations.

THURSDAY

• BD™ Cytometric Bead Array (CBA) – Overview of Principles, BD™ Cytometric Bead Array Software , Applications.
• Neutrophil Function – Phagocytosis (PHAGOTEST®) and Oxidative burst (BURSTTEST/PHAGOBURST®).
• Basophil - Allergy Testing by Flow Cytometry (BASOTEST®).

FRIDAY

• Post-Acquisition Data Manipulation, Compensation, Statistical Analysis and Presentation of Data.
• FlowJo Software.
• Good Laboratory Practices (GLP) and Flow Cytometry.
• Operational Troubleshooting and Maintenance.
• Review and Discussion of Course II.

Part III Research Applications in Flow Cytometry

MONDAY

• Cell Viability – Propidium Iodide (PI) Exclusion.
• DNA Analysis, Cell Cycle Analysis and S-Phase Determination, DNA Ploidy and QC using ModFit LT™ Software (Verity Software House) .
• Cell Proliferation - Ki67, BrdU Incorporation.

TUESDAY

• Apoptosis - Annexin-V, Caspase-3 and Cleaved PARP.
• Mitochondial Markers for Apoptosis – Cytochrome c Release (BD™ MitoScreen)
• DNA Fragmentation - Tunnel Assay using APO-Direct™ (Phoenix Flow Systems)

WEDNESDAY

• Cell Function – Intracellular pH and Ca++ Flux
• Cell Signalling – Phosphorylation (BD Phosflow™)
• Cytometric Bead Array (CBA) – Human Th1/Th2 Cytokine Kit using BD FACSArray™ and BD™ Cytometric Bead Array Software software (CBA cytokine)

THURSDAY

• Rare Event Analysis - Peripheral Blood Stem Cell Enumeration – BD Procount™ System (IVD) (Progenitor Cell (CD34/CD45) Enumeration Kit)
• Peripheral Blood Dendritic Cells Identification (BD™ Dendritic cell reagents).
• Statistical Analysis of Rare Events

FRIDAY

• Cell Proliferation Tracing using Carboxy Fluorescein Diacetate Succinimidyl Ester (CFSE) and ModFit LT™ Software (Verity Software House).
• Bacterial Viability, Monitoring and Enumeration.
• Review and Discussion of Course III.