Hello Prachi,

You should treat the cells 24 hours after plating them in 96-well plate.

This time is often chosen because by 24 hours, many cell types have typically reached the exponential growth phase in which cells are dividing rapidly and have reached their maximum growth rate. This is a good time to apply treatment because the effects of the treatment will be seen more quickly and directly on the actively dividing cells. 

After plating, the cells will need some time to attach to the surface of the well and begin to establish themselves in the new environment. This phase which is called the attachment phase takes around 24 hours, after which the cells start their exponential growth, which is the right time to treat the cells.

So, plate SH-SY5Y cells once the chemical arrives. The present 96-well plate which you have seeded may be discarded because it is just a waste of time maintaining them in 96-well plate, assuming that you are already maintaining them in flasks.

Best,

You can grow your cells in the 96-well until you get the confluence percentage you need, granted you change your medium frequently. It is recommended that you seed your cells overnight (+/- 24 hours) for 2D cultures, however, the important thing is to be consistent

Prachi Kul

Hi!

SH-SY5Y cells seeded at 20,000 cells/well in a 96-well plate can typically be maintained for 48–72 hours under standard culture conditions (37°C, 5% CO₂) without significant overgrowth or nutrient depletion, provided the media volume (~100–200 µL/well) is optimal and refreshed every 48 hours if extended culture is needed. However, their doubling time is ~48 hours, and after 72 hours post-seeding, they may reach high confluency, leading to changes in morphology, gene expression, and response to treatments. For best experimental reproducibility, it is advisable to either treat within 24–48 hours or re-plate fresh cells once your chemical arrives, especially if cell cycle, viability, or differentiation status is critical to your assay.

The optimal incubation time for cells in a 96-well plate before treatment typically ranges from 24 to 48 hours, depending on the cell type and experimental conditions. For most adherent cell lines, a 24-hour incubation is sufficient to allow proper attachment and recovery after seeding, ensuring they reach an appropriate confluency (usually 60–80%) before treatment. Slow-attaching or sensitive cells, such as primary cells or neurons, may require a longer incubation period of 48–72 hours to fully adhere and stabilize. Suspension cells, on the other hand, often need only 4–24 hours before treatment since they do not require attachment. The exact timing should be determined based on cell growth rate, seeding density, and assay requirements. It is essential to microscopically confirm cell adherence and health before proceeding with treatment. If cells have undergone stressful procedures like thawing or transfection, extending the recovery time to 48 hours may improve experimental consistency. Always optimize incubation time for your specific cell line and assay to ensure reliable results. Let me know if you need guidance for a particular cell type or application.