The cell detachment procedure is a critical step in cell culture for subculturing, harvesting cells, or performing downstream experimental applications. The choice of detachment method depends on the cell type, culture conditions, and the specific experimental requirements. Here is a general procedure for enzymatic cell detachment using trypsin-EDTA, which is commonly used for adherent cell lines:

1. Prepare materials: Ensure that all required materials are available, including trypsin-EDTA solution, sterile PBS (phosphate-buffered saline), culture media, a serological pipette, a pipettor with sterile tips, a centrifuge tube, and an inverted microscope.
2. Aspirate the media: Using a serological pipette, carefully aspirate the cell culture media from the culture vessel without disturbing the adherent cells.
3. Wash the cells: Gently add sterile PBS to the culture vessel to wash the cells and remove any residual culture media. Aspirate the PBS, taking care not to disturb the cells.
4. Add trypsin-EDTA solution: Add an appropriate volume of pre-warmed trypsin-EDTA solution to the culture vessel, ensuring that the cell monolayer is completely covered. Incubate the cells with trypsin-EDTA at 37°C for 1-5 minutes, depending on the cell type and the extent of cell confluency. It is essential to closely monitor the cells under an inverted microscope to avoid over-trypsinization, which can damage the cells.
5. Check for cell detachment: Observe the cells under an inverted microscope to confirm that they have detached from the surface and are rounding up. Gently tap the side of the culture vessel to facilitate the detachment of any remaining cells.
6. Neutralize trypsin activity: Add an equal volume of culture media containing serum or a trypsin inhibitor to the culture vessel to neutralize the trypsin activity and prevent damage to the cells.
7. Collect and transfer cells: Gently mix the cell suspension to ensure a homogeneous distribution of cells, and transfer the cell suspension to a centrifuge tube.
8. Optional centrifugation step: If necessary, centrifuge the cell suspension at a low speed (e.g., 1000 rpm) for 3-5 minutes to pellet the cells. Remove the supernatant and resuspend the cell pellet in fresh culture media.
9. Subculture or proceed with downstream applications: Depending on your experimental requirements, either subculture the cells into new culture vessels, counting the cells if needed, or proceed with downstream applications such as flow cytometry, cell-based assays, or RNA/DNA isolation.

Remember to follow appropriate sterile techniques and guidelines specific to your cell line and laboratory protocols.