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
- 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.
- Count and prepare 5-10 million cells in an eppendorf tube. Pellet and remove the supernatant.
- 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 CellsIsotype: 100µl Cells + 20µl (1 test) Ms IgG AF647/ APCPax6 Sample: 100µl Cells + 5µl (1 test) of AF647-mouse-anti human Pax6
- Incubate tubes at room temperature in the dark for 30 minutes.
- Pellet then wash each tube 2x with 1ml 1x BD Permwash buffer. (centrifuge 4 minutes at 200 x g).
- Resuspend pellets in 1x DPBS 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.
- Scale against unstained control and perform an analysis gating negative population from the isotype control.