you can deploy Copilot Separating migratory and non-migratory cells in a Boyden Chamber Assay can be a bit tricky, but here are some steps and tips to help you out:

### Steps to Separate Migratory and Non-Migratory Cells

1. **Prepare the Boyden Chamber:**

- **Upper Chamber:** Place your cell suspension in the upper chamber.

- **Lower Chamber:** Fill the lower chamber with a medium containing chemoattractants to encourage cell migration.

2. **Incubation:**

- Allow the cells to incubate for the appropriate time to enable migration through the membrane.

3. **Collecting Migratory Cells:**

- **Remove the Upper Chamber:** Carefully remove the upper chamber without disturbing the lower chamber.

- **Wash the Membrane:** Gently wash the membrane to remove any non-migratory cells that might be loosely attached.

- **Scrape the Membrane:** Use a cell scraper to collect the migratory cells from the lower side of the membrane. Alternatively, you can use trypsin to detach the cells.

4. **Collecting Non-Migratory Cells:**

- **Upper Chamber Cells:** Collect the non-migratory cells that remain in the upper chamber by gently pipetting them out.

### Tips for Reliable Separation

- **Gentle Handling:** Be gentle when handling the cells to avoid disrupting the membrane or causing cell damage.

- **Optimal Pore Size:** Ensure that the pore size of the membrane is appropriate for your cell type. Typically, a pore size of 8 µm is suitable for most cell types.

- **Staining:** Stain the cells to differentiate between migratory and non-migratory cells. This can help in visualizing and counting the cells accurately.

- **Validation:** Use complementary techniques like flow cytometry or microscopy to validate the separation and ensure the purity of the collected cell populations.

### References

- **[Boyden Chamber Technique](https://www.emdmillipore.com/US/en/life-science-research/antibodies-assays/assays-overview/cell-invasion-migration-assays/boyden-chamber-technique/I0qb.qB.KSMAAAFANtY.1ZcQ,nav)**

- **[Cell Migration and the Boyden Chamber](https://link.springer.com/protocol/10.1385/1-59259-137-X:047)**

- **[Discussion of Cell Migration Assay Formats](https://www.cellbiolabs.com/news/discussion-cell-migration-assay-formats)**

These steps and tips should help you effectively separate migratory and non-migratory cells in your Boyden Chamber Assay.

Good luck