Protocol Definitive Endoderm Marker Sox17 Staining for Flow Analysis
Protocol for Sox17 Staining for FACS Analysis
Primary Antibody : BD 561590 : mouse anti-human Sox17 (0.5mg/ml) (use at 1:500)
Secondary Antibody : Invitrogen A10680 : AF488-Goat anti-human IgG IgM (H+L) 2mg/ml (use at 1:400)
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 BD Permwash buffer.
  7. Resuspend each sample in 300µl of 1x BD Permwash buffer and divide equally into 3 tubes.
  8. Label and prepare tubes as below: Unstained, Isotype, Pax6 Sample
    Unstained: 100µl Cells
    Seconday Only: 100µl Cells
    Sox17 Sample: 100µl Cells + 0.5µl Primary antibody
  9. Incubate tubes at room temperature for 30 minutes.
  10. Pellet then wash each tube 2x with 1ml 1x BD Permwash buffer. (centrifuge 4 minutes at 200 x g).
  11. Resuspend Secondary only and Sox 17 sample in 100µl BD Staining Buffer (with FBS) cat# 554656.
  12. Add 0.25µl of secondary antibody to each tube and incubate in the dark at room temperature for 1 hour.
  13. Pellet and wash each tube 2x with 1x DPBS
  14. Resuspend each pellet to a final volume of 300µl (200µl for small pellets or low cell counts)
  15. 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.