I am working with triple negative breast cancer cell line MDA-MB-231. I want to evaluate the effects of a specific human rrecombinant protein on them and to evaluate cytokine production in supernatants by ELISA assay. I am starting now to perform this experiments and I don't know which kind of plate to choose (12, 24, 48 wells) and what number of cells to start from with a good confluence. Could you help me?
Hello Simone Marcella
For such an assay, I would use 96-well plate. You will have to plate a smaller number of cells and the maximum volume of media per well for a 96-well plate would be around 250-300ul.
Since you are not aware of the seeding density, before you begin your experiments, I would suggest you perform a pilot experiment to decide on the seeding density. You may plate different cell densities say from a minimum of 10000 to a maximum of 25000 cells per well for a 96-well plate depending on cells’ doubling time. Decide on the seeding density that will give you 80% confluency on the day of treatment as overconfluent cells may give you erroneous results.
Good Luck!
@all When selecting the type of plate and the initial number of cells for your experiments with MDA-MB-231 cells, consider the following factors:
Based on these considerations, here are some general guidelines:
- For initial experiments, a 24-well plate is often a good starting point. This allows for multiple conditions, replicates, and easy handling of the cells.
- Seed the cells at a suitable density to achieve the desired confluency. For MDA-MB-231 cells, seeding 50,000 to 100,000 cells per well in a 24-well plate can be a reasonable starting point. However, the optimal density may vary based on your specific experimental requirements and the growth characteristics of the cells. It is advisable to perform a pilot experiment to optimize the seeding density if needed.
- Monitor the cells regularly under a microscope to assess their growth and confluency. Typically, MDA-MB-231 cells reach 70-80% confluency within 24-48 hours after seeding, but this can vary depending on culture conditions and the experimental setup.
Remember to follow the standard cell culture protocols for maintaining MDA-MB-231 cells, including appropriate media, supplements, and sterile techniques. Additionally, consult any specific guidelines provided by the supplier or literature for the recombinant protein you are using to ensure proper experimental conditions.
As you perform the experiments and gain experience with your specific system, you can fine-tune the cell seeding density and plate size based on your experimental requirements and desired outcomes.
Ahmad Al Khraisat AND Malcolm Nobre thank you very much for your replies. My doubt is, if I have to seed 50000-100000 cells/well in a 24 well and MDA-MB-231 doubling time is of 24-48h to reach a 70-80% confluency, must I leave cells reach the confluency before to stimulate with human recombinant protein or must I seed the cells with already an high confluency and soo stimulate with recombinant protein? Thank you very much
Simone Marcella @all You're welcome! I understand your question regarding the seeding and stimulation of MDA-MB-231 cells with human recombinant protein.
To determine the optimal experimental conditions, it's essential to consider the growth rate of MDA-MB-231 cells and your specific experimental requirements. Here are two scenarios you can consider:
Ultimately, the decision between these two approaches will depend on your specific experimental goals and the nature of the study you are conducting. Both approaches have their advantages and could provide valuable insights into the cellular response to the human recombinant protein. Additionally, it's essential to consider the specific requirements of your downstream assays or analyses, as different experimental conditions could influence the outcomes.
Before starting the experiments, it's always a good idea to perform some pilot studies to optimize the cell seeding density and timing of stimulation to ensure you achieve the desired experimental conditions for your study.
Dear,
The number of cells is depend on perticular assay which you want to perform. Examples, if you want to perform Scratch assay then you need more number of cells and if you wish to do colony formation assay then you need less number of cell (600 cells per well in 6-well culture plate).
You can visist Manufacturer company of your culture plates regarding this.. they provide cell number on thier websites. refere cell number to respective culture plates.
Third, you can refer literature where you can find cell number for respective culture plates and fourth, you can optimise yourself. When diameter of plate is decrease, cell number will be also decrease.
As of my concern, I used to seed 7 thousands, 30 thousands, 50 thousands and 1 lacs cells per well in 96, 48, 24 and 12 well plates respectively.
Thank you
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