Presentation and demonstration of flow cytometers

9:00 am – noon
Thomas Kamradt / Nico Andreas
University Hospital Jena, Immunology

9:00 am – 10:30 am
Presentation about handling of flow cytometers

  • general¬†information
  • choice of fluorochromes (e.g. 20% overlap of 10-fold brighter fluorochrome on a dim fluorescence)
  • steps to set up a machine for multi-colour stainings (first FSC-SSC, second full mix, acquisition, test of FSC by gating on target pops, proper setup of the machine)
  • compensation (beads, single cells, by eye, DAPI and APC-Cy7)
  • how to produce comparable results by using two different machines (use of CS&T beads mean fluorescence)
  • experimental tips:
    – CFSE patterns (only works nicely on naive T cells)
    – ery lysis for FoxP3 staining (no ery lysis needed)
    – intracellular cytokine staining (ON in PBA-E)

11:00 am – noon
Practical demonstration and questions

Demonstration of FACS analysis using a Canto Plus flow cytometer (BD Bioscience)

  • Spleen samples prepared:
    – mixed staining: CD19, CD4, CD8, TCRb, CD11c, CD11b, DAPI
    – single stainings for each fluorochrome
    – non-stained control
  • bead-assisted compensation in comparison to single cell stainings

 

Map

Institute of Immunology
Leutragraben 3
07743 Jena