What is a cell seeding protocol?

Cell seeding is usually the first protocol step and a standard procedure in cell-based experiments. A correct and standardized cell seeding protocol is a critical factor for reproducible experimental results. The main challenge in this step is to achieve and maintain comparable cell numbers in all repeated experiments.

How do I start a cell seeding process?

Here is the correct way to conduct the cell seeding process: Get all of your supplies ready. You'll need your cryopreserved vial, a beaker or water bath filled with pre-warmed water, and a flask with pre-equilibrated media.

Should I resuspending or mixing a cell seed?

So, although resuspending or mixing is necessary, you should not overdo it and avoid too much pipetting to reduce air bubble formation. Gentle pipetting also reduces shear forces and stress for your cells – a critical part of every cell seeding protocol. Image 2: Air bubbles formed during cell seeding can hinder cell attachment.

Why is cell sedimentation important in cell seeding?

Image 1: Cell sedimentation is a critical factor to consider in every cell seeding protocol. Cells sediment in the vessel within minutes – the longer the cells seeding process takes, the lower the number of cells in the supernatant. The process of cell sedimentation is quite fast, within minutes.

What happens if you prefill a vessel with cell culture medium?

On the left, you see what happens if you just prefill the vessel with cell culture medium, add the cells, and call it a day. As a result, the cells mainly adhere in the middle of the vessel. To achieve an equal cell distribution, some people use a “figure-eight” movement, while others prefer a cross-like movement of the plate or dish.

How do you fix a cell culture medium?

Discard the cell culture medium by inverting the slide and gently tapping it on a paper towel to remove the remaining medium. Fix the cells with 4% formaldehyde (diluted in 1X PBS— prepare fresh) for 10 min at room temperature (fixation time can be increased to 20 min depending on the cell line). Fixed cells can be stored at 4°C for up to 1 week.