Hello Sara Sultana

Try gradually introducing the fixative by adding a small amount of 4% PFA (for instance 500ul) to the existing growth medium, wait for two minutes, then aspirate it and apply the pure 4% PFA. The abrupt change in osmolarity between the culture medium and the PFA solution can cause the cells to detach.

Please note that HCT116 cells do have some unique characteristics that require attention during the protocol to prevent potential issues like cell detachment and clumping.

1. HCT116 cells can be sensitive, so handle them carefully during the fixation process to maintain cell integrity and proper attachment to the coverslip.

2. HCT116 cells are adherent but have lower adhesion strength than some other cell lines.

3. HCT116 cells have less contact inhibition than other cell types and can grow in layers, forming clumps if they become overconfluent. This leads to uneven fixation and poor staining.

4. For IF assays, aim for a subconfluent cell density (typically 50–70%) at the time of fixation. This ensures a uniform monolayer of cells on the coverslip.

5. Preparing fresh PFA fixative immediately before use is often recommended for consistent results.

Best,

Some considerations before PFA fixation.

1. Cell attachment to glass cover slips. before starting the fixing process, ensure the cells are firmly attached to the glass coverslip (By observing the morphology).

2. Wash any residual media. Provide PBS wash to the cells (x3times) and observe if cells are getting detached. If the cells are firmly attached, proceed with the fixation step.

3. Confluency of cells: Maintain at around 60-70%