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Protocol Neuroectoderm Marker PAX6 Staining for Flow Analysis
Fixed Nuclear Marker Staining for Flow Analysis
Using AF647-mouse-anti human Pax6 (BD Cat: 562249) as an example
  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. Count and prepare 5-10 million cells in an eppendorf tube. Pellet and remove the supernatant.
  5. Resuspend each pellet using 1ml BD Cytofix buffer (contains 4.2% Paraformaldehyde).
  6. Incubate cell suspension at room temperature for 20 minutes. Cells are now fixed.
  7. Pellet and wash fixed cells 2x with 1ml 1x BD Permwash buffer.
  8. Resuspend each sample in 300µl of 1x BD Permwash buffer and divide equally into 3 tubes.
  9. Label and prepare tubes as below: Unstained, Isotype, Pax6 Sample
    Unstained: 100µl Cells
    Isotype: 100µl Cells + 20µl (1 test) Ms IgG AF647/ APC
    Pax6 Sample: 100µl Cells + 5µl (1 test) of AF647-mouse-anti human Pax6
  10. Incubate tubes at room temperature in the dark for 30 minutes.
  11. Pellet then wash each tube 2x with 1ml 1x BD Permwash buffer. (centrifuge 4 minutes at 200 x g).
  12. Resuspend pellets in 1x DPBS to a final volume of 300µl (200µl for small pellets or low cell counts)
  13. 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.
  14. Scale against unstained control and perform an analysis gating negative population from the isotype control.