Cell culturing refers to the process of growing and maintaining cells under controlled conditions in a laboratory setting. A basic cell culturing protocol involves several steps, including cell line selection, medium preparation, passaging, and maintaining the proper growth conditions. Here is a general outline of a cell culturing protocol:

1. Cell line selection: Choose the appropriate cell line for your experiment based on its characteristics, such as origin, growth properties, and susceptibility to specific treatments.
2. Thawing frozen cells: If you are starting with frozen cells, thaw them quickly in a 37°C water bath and transfer them to a culture medium to dilute the cryoprotectant. Centrifuge the cells to remove the cryoprotectant and resuspend the cell pellet in fresh culture medium.
3. Medium preparation: Prepare the appropriate growth medium for your cell line, typically composed of a basal medium supplemented with various factors such as serum, growth factors, hormones, and antibiotics. Sterilize the medium using a 0.22 μm filter and store it at 4°C.
4. Plating cells: Transfer the resuspended cells into a tissue culture flask or dish containing the prepared medium. Incubate the cells in a humidified incubator at 37°C with 5% CO2 (standard conditions for most mammalian cell lines).
5. Monitoring cell growth: Monitor the cells daily under an inverted microscope, checking for confluency (the percentage of surface area covered by cells) and any signs of contamination.
6. Passaging cells: Once the cells reach 70-90% confluency, passage them to prevent overgrowth and maintain a healthy culture. To passage cells:a. Aspirate the old medium from the flask or dish. b. Wash the cells gently with phosphate-buffered saline (PBS) to remove any residual medium. c. Add an appropriate amount of trypsin-EDTA solution to detach the cells from the surface. d. Incubate the cells at 37°C for a few minutes until they detach (monitor under a microscope). e. Add fresh culture medium to neutralize the trypsin, and gently pipette the cell suspension up and down to break up cell clumps. f. Transfer the cell suspension to a new flask or dish with fresh medium, or centrifuge and resuspend in fresh medium if necessary. g. Incubate the cells under standard conditions and continue to monitor growth.
7. Cryopreservation: If you need to store cells for future use, cryopreserve them by resuspending the cell pellet in a freezing medium (usually culture medium with 10% DMSO and 50% serum). Transfer the cell suspension to cryovials and place them in a controlled-rate freezing container, which is then stored at -80°C overnight before transferring the vials to a liquid nitrogen tank for long-term storage.
8. Aseptic technique: Throughout the entire cell culture process, maintain aseptic conditions to prevent contamination. Work in a sterile biosafety cabinet, use sterile equipment, and practice proper handling techniques.

This general protocol can be adapted based on the specific requirements of your cell line or experimental design. Always consult the cell line’s datasheet or relevant literature for optimal growth conditions and any special considerations.