I am plating 1 million mouse spleen cells in 96-well plates. In different wells, I am adding different proteins at a concentration of 1 ug/well to try to stimulate T-cell activity. I will then incubate the spleen cells + protein for 72 hours in standard conditions (37C, 5% CO2). At the end of the 72h incubation, I plan to collect supernatant for downstream cytokine analysis. What 96-well plate bottom shape is best for this application?
I have previously performed this experiment in V-bottom wells because I needed to centrifuge them at the end and separate cells from supernatant. However, I am concerned that the V-bottom wells didn't provide enough surface area for the protein to interact with the spleen cells, since the spleen cells pelleted on the bottom after some time. Perhaps I should have incubated the cells and protein in flat bottom wells and then transferred them to v-bottoms right before centrifugation. Additionally, during the 72h incubation period, would it be beneficial to mix the cells and proteins with a multichannel one or more times, just to increase cell and protein interaction?
Hi Gage,
I have extensive experience incubating mouse spleen cells for cytotoxicity assays from my PhD work. Based on that experience, I highly recommend using flat-bottom plates instead of v-bottom plates. Here's why:
- Better cell health and viability: Spleen cells tend to settle comfortably at the bottom of flat-bottom plates, especially after periods of inactivity like overnight incubation. This minimizes stress and promotes their well-being. If you leave them unagitated for more than an hour, most cells will remain happily resting on the bottom surface. You can even do a test run by centrifugation of flat-bottom plates before your actual experiment to confirm this.
- Improved cellular respiration: In v-shaped plates, cell accumulation at the bottom can limit efficient gas exchange and nutrient access. This can restrict cellular respiration and potentially lead to cell death, which you understandably want to avoid.
Best of luck with your experiment!
Ali
Hello Gage,
For your application, you will need to consider the growth support topography and growth culture conditions in terms of surface area and volume of culture medium available to the cultured cells.
The V-bottom wells are not ideal for your application because as you mentioned, it will not provide enough surface area for the protein to interact with the spleen cells, since the cells will settle at the bottom after some time and cell activity will be significantly lowered. Moreover, in V-bottom wells, the cell morphology will also be affected as cells will appear to aggregate in the centre of the well. This is more relevant if cells are adherent in nature.
I agree to what you mentioned regarding incubating the cells and protein in flat bottom wells and then transferring them to V-bottom wells right before centrifugation.
During the 72h incubation period, do not mix the cells and proteins with a multichannel one or more times because this action of yours will disturb the interaction between the cells and the environment, and you will be doing more harm than good. Cell and protein interaction will occur within the wells due to the Brownian motion of protein molecules. You need not worry about that.
The article attached below will be helpful!
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662085/
Best.
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