??Division of Immunology

?SU Immunology Research Group (SU-IRG)

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?PLATFORMS

Flow Cytometry

Flow cytometry is an analytical technique that utilizes light to count and profile cells in a heterogeneous fluid mixture. It is a particularly powerful method because it allows a researcher to rapidly, accurately, and simply collect data related to many parameters from a heterogeneous fluid mixture containing live cells. For example, in immunology flow cytometry is used to identify, separate, and characterize various immune cell subtypes by virtue of their size, morphology, surface phenotype and intracellular production of immune mediators. As such, flow cytometry has numerous applications in science, including those relevant to healthcare. The technology has been widely used in the diagnosis of health conditions, particularly diseases of the blood such as leukaemia, although it is also commonly used in the various different fields of clinical practice as well as in basic research and clinical trials.

While conventional flow cytometry uses the one detector, one fluorochrome approach, spectral flow cytometry makes use of all detectors in the cytometer to capture a signature of the full emission spectra of each fluorochrome. Therefore, we are able to distinguish fluorochromes with a similar emission spectra. Imaging flow cytometry combines the high-event-rate nature of flow cytometry with detailed imaging properties of microscopy. The measurement of large numbers of features from the resulting images provides rich data sets that have resulted in a wide range of novel biomedical applications.

Our highly trained staff offers a wide range of expertise in:

  • Study and panel design as well as discussing gating strategies for multi-colour experiments.
  • Processing of different sample types such as whole blood cells, PBMCs, cultured cells and cell lines, pericardial fluid, bacteria.
  • Acquiring of samples using different Flow Cytometers:
    • Imaging flow cytometer – Amnis ImageStreamX: 8 laser (375nm, 405nm, 488nm, 561nm, 592nm, 642nm, 730nm, 785nm), 12 channels
    • Conventional Flow Cytometer – Beckman Coulter DxFlex:
    • 3-laser (405nm, 488nm, 638nm), 13 channels: Spectral Flow Cytometer – Cytek Aurora
    • 4 laser (405nm, 488nm, 561nm, 640nm), 48-channel with capability of autofluorescence extraction.
    • Cell Sorter – Beckman Coulter CytoFlex SR: 4 laser (405nm, 488nm, 561nm, 638nm), 15 channels
  • Analysis of samples using FlowJo.

 

Our staff is experienced in the following applications:

  • Immunophenotyping of whole blood or cells for innate and adaptive cell frequencies and subsets, including maturation, trafficking, activation, proliferation and chemokine and cytokine expression.
  • Determining the percentages and absolute counts of T, B, and natural killer (NK) cells as well as the CD4 and CD8 subpopulations of T cells in peripheral blood using BD Multitest? 6-color TBNK reagent and BD Trucount? tubes.
  • Live/ dead discrimination of bacteria.
  • Infectious and non-infectious cell sorting of cells and bacteria.

If you consider using Flow cytometry and need guidance in setting up your experiments, panel design, acquiring and analysis of your samples; contact our Flow Cytometry expert  Andrea Gutschmidt.

 

Luminex

Luminex is a dual-laser flow-based detection technology and allows for analysis of up to 500 analytes in a single well. Our staff has vast experienced in polystyrene or magnetic beads-based assays. Amongst the multiplex biomarker analysis platforms currently in our lab are a Luminex 200 (can assay up to 100 targets per well), and IntelliFlex (up to 500 targets per well) instruments. We can conduct Luminex experiments in both 96-well and 384-well plates and have installed a Curiox Drop Array system to complement our Luminex offering In addition to the Luminex instruments, we also have a Meso Scale Discovery (MSD) SQ120 instrument. ?

If you wonder if Luminex (or MSD) is the right platform for your assays, and if you strive for accuracy and reproducibility speak to our Luminex experts Mrs Candice Snyders and Prof Novel Chegou.

 

Seahorse XFp Analyzer

The Agilent Seahorse Extracellular Flux (XF) Analyzer is the world's most powerful metabolic analyzer. It is based on measuring rates of change in pH and Oxygen concentration in the two metabolic pathways by which cells use to generate energy, i.e. Glycolysis and Oxidative Phosphorylation respectively. It offers the opportunity for functional metabolic analysis essential to tell the full story of cellular processes and pathologies in real time within minutes. The Seahorse technology enables the measurement of metabolic function and energy phenotype in patients-derived, and other precious samples. ?

We run samples in our lab using the Agilent Seahorse XFp Analyzer. For clinical appraisal of cellular energy metabolism, the following questions can be answered using the Seahorse analyzer:

  • XF Cell Energy Phenotype Test, measure energy phenotype and possible metabolic switches:
    • Define XF Metabolic Phenotypes (Glycolysis or Mitochondrial Respiration)
    • Measure XF Metabolic Potentials (Cells ability to meet up with its energy demands)
    • Study Metabolic Switches (From Glycolysis to Oxidative phosphorylation and vice versa)
  • XF Glycolysis Stress Test, measure the key parameters of glycolytic function:
    • Glycolysis
    • Non-glycolytic Acidification
    • Glycolytic Capacity 
    • Glycolytic Reserve
  • XF Mitochondrial Fuel Flux Test, measure the key parameters of mitochondrial fuel use:
    • Fuel Dependency
    • Fuel Flexibility
    • Fuel Capacity
  • XF Cell Mitochondrial Stress Test, measure the key parameters of mitochondrial function:
    • Basal Respiration
    • ATP Production
    • Maximal Respiration and Spare Capacity
    • Proton Leak
  • Study metabolic fluxes of cells treated with different drugs through injection ports

If you consider using the Seahorse technology for metabolic analysis, contact our specialist  Prof Nelita du Plessis.?