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)
- 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 DPBS.
- Resuspend each sample in 300µl of 1x R&D Permwash (FC004) buffer and divide equally into 3 tubes.
- Label and prepare tubes as below: Unstained, Isotype, Pax6 Sample
Unstained: 100µl CellsIsotype Control: 100µl Cells + 10µl Goat IgG Control-APCBrachyury Sample: 100µl Cells + 10µl anti-h/m Brachyury-APC pAb IgG- APC
- Incubate tubes at room temperature for 30 minutes.
- Pellet then wash each tube 2x with 1ml 1x R&D Permwash (FC004) (centrifuge 4 minutes at 200 x g).
- Resuspend each pellet to a final volume of 300µl in 1x DPBS (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.