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Protocol Mesoendoderm Marker Brachyury Staining for Flow Analysis
Protocol for Brachyury Staining for Fixed Flow
Antibody: R&D Systems Goat anti-h/m Brachyury-APC Cat: IC2085A 10µl/test
Isotype Control: R&D systems Goat IgG Control-APC Cat: IC108A 10µl/test
Note: It is recommended to prepare 1-5 million cells per cell type (3 wells of a 24 well plate, 2 wells of a 12 well plate or 1 well of a 6 well plate)
  1. Detach cells of interest from the culture dish. For hESCs use Accutase.
  2. Aspirate media off cells in culture dish and add Accutase.
  3. Incubate at 37ºC for 30 minutes. Collect the cells in the Accutase suspension. Pellet (centrifuge 4 minutes at 200 x g) and remove the accutase.
  4. Resuspend each pellet using 1ml BD Cytofix buffer (contains 4.2% Paraformaldehyde).
  5. Incubate cell suspension at room temperature for 20 minutes. Cells are now fixed.
  6. Pellet and wash fixed cells 2x with 1ml 1x DPBS.
  7. Resuspend each sample in 300µl of 1x R&D Permwash (FC004) buffer and divide equally into 3 tubes.
  8. Label and prepare tubes as below: Unstained, Isotype, Pax6 Sample
    Unstained: 100µl Cells
    Isotype Control: 100µl Cells + 10µl Goat IgG Control-APC
    Brachyury Sample: 100µl Cells + 10µl anti-h/m Brachyury-APC pAb IgG- APC
  9. Incubate tubes at room temperature for 30 minutes.
  10. Pellet then wash each tube 2x with 1ml 1x R&D Permwash (FC004) (centrifuge 4 minutes at 200 x g).
  11. Resuspend each pellet to a final volume of 300µl in 1x DPBS (200µl for small pellets or low cell counts)
  12. At this stage cells can be transferred to round bottom cytometer tubes for immediate analysis. If not analyzing immediately, store samples in eppendorf tubes protected from light, for up to 5 days before analyzing.