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)
- Detach cells of interest from the culture dish. For hESCs use Accutase.
- Aspirate media off cells in culture dish and add Accutase.
- 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.
- Resuspend each pellet using 1ml BD Cytofix buffer (contains 4.2% Paraformaldehyde).
- Incubate cell suspension at room temperature for 20 minutes. Cells are now fixed.
- Pellet and wash fixed cells 2x with 1ml 1x BD Permwash buffer.
- Resuspend each sample in 300µl of 1x BD Permwash buffer and divide equally into 3 tubes.
- Label and prepare tubes as below: Unstained, Isotype, Pax6 Sample
Unstained: 100µl CellsSeconday Only: 100µl CellsSox17 Sample: 100µl Cells + 0.5µl Primary antibody
- Incubate tubes at room temperature for 30 minutes.
- Pellet then wash each tube 2x with 1ml 1x BD Permwash buffer. (centrifuge 4 minutes at 200 x g).
- Resuspend Secondary only and Sox 17 sample in 100µl BD Staining Buffer (with FBS) cat# 554656.
- Add 0.25µl of secondary antibody to each tube and incubate in the dark at room temperature for 1 hour.
- Pellet and wash each tube 2x with 1x DPBS
- Resuspend each pellet to a final volume of 300µl (200µl for small pellets or low cell counts)
- 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.