It sounds like you're encountering some challenges with the adherence and clumping of Caco-2 cells in inserts. Here are some tips that might help improve your cell attachment and spread:

1. **Coating the Inserts:**

- Consider coating your inserts with extracellular matrix proteins (like collagen, fibronectin, or Matrigel) to enhance cell adhesion. This is especially important for Caco-2 cells, as they may require a conducive surface to adhere properly.

2. **Cell Seeding Density:**

- Adjust the seeding density. Sometimes, a lower density (e.g., 50,000–100,000 cells per insert) can help cells spread more uniformly and reduce clumping.

3. **Media Composition:**

- Ensure that the media you use is suitable for cell attachment. You could try using media without antibiotics initially to minimize any stress on the cells. Additionally, consider adjusting the FBS concentration, as too high or too low can affect adhesion.

4. **Gentle Handling:**

- Be gentle during the resuspension of cells and while transferring them to the inserts. Harsh pipetting can lead to cell clumping.

5. **Incubation Conditions:**

- Check the incubation conditions, such as temperature and CO2 levels. Caco-2 cells thrive in a 37°C, 5% CO2 environment. Ensure that the inserts are placed in the correct environment promptly after seeding.

6. **Cell Passage:**

- If the cells are consistently problematic, consider using cells from a different passage or re-culturing them from a frozen stock. Sometimes, older passages can lose their adherence properties.

7. **Pre-warming Inserts:**

- Pre-warm the inserts and media before seeding to ensure that temperature changes do not affect cell behavior.

8. **Physical Disturbance:**

- Minimize any disturbances after seeding. Allow cells to settle and adhere without movement for a few hours before handling them again.

If you've already tried these suggestions and are still having issues, it may be worth consulting specific protocols or publications that address Caco-2 cell culture in inserts for any additional techniques that could be beneficial.

You can also reach out to fellow researchers or forums for Caco-2 cells, such as **ResearchGate** or **the Cell Culture section of the American Society for Cell Biology**, where you might find more tailored advice based on shared experiences.

Hi Samanvita,

Make sure you are seeding the cells at an appropriate density. Have you tried different concentrations? if not, for inserts you can include a condition with a cell density of 2x10^5 cells/cm². Also, ensure that the inserts are properly hydrated before seeding; pre-treating the membrane with media or PBS for at least 30 min can help (and make sure the inserts aren't too dry when adding the cells, as this can negatively affect adhesion). Sometimes, coating the inserts with collagen, fibronectin (e.g., 40μg/mL), or gelatin can improve cell attachment. Good work for your experiments!