Hi everyone, I'm working with PANC-1 cells and experiencing a problem with cell survival after passaging. I passaged my cells from a confluent T25 flask into two six well plate and rest of it to T25 flask, but a few days later most of (in T25 flask) the cells started dying.
I suspect that I seeded too few cells for such surface area. The attached image shows the cell density after a few days of seeding in the T25 flask.
I would appreciate any advice on:
- Minimum recommended seeding density for PANC-1 in T25 flasks
- Whether low density alone could explain such cell death
- Any tips to improve cell attachment and survival after splitting
Thanks in advance!
Hello,
Thank you for your answer Philippe Paget-Bailly
. Here are the details.I seeded two 6-well plates using cells from a confluent T25 flask — approximately 1.5 million cells in total. After 48 hours, I performed protein isolation from the 6-well plates following treatment.
In parallel, I passaged some of the same cells into a new T25 flask at approximately a 1:6 dilution ratio. I only added fresh medium (high glucose DMEM with 10% FBS and 1% pen/strep) every two days, but after about a week, the cells in the new T25 flask still haven't recovered or proliferated well.
I’ve also noticed that when the seeding density is low, small dark particles appear in the spaces between cells. These resemble cellular debris or possible precipitates, but I’m not sure of their exact nature.
Any insights or suggestions would be appreciated!
Hello Bahar Sarikamis Johnson,
PANC-1 cells are known to be sensitive to seeding density, meaning the initial number of cells plated can significantly impact their growth and behavior.
For PANC-1 cells, a common seeding density is 0.5 - 1 x 10^5 cells/cm2. To calculate the total number of cells needed, multiply the seeding density by the growth area: (0.5 - 1 x 10^5 cells/cm2) * (25 cm2) = (1.25 - 2.5) x 10^6 cells. This comes to approximately 1 x 10^6 cells per T-25 flask.
You passaged some of the same cells into a new T-25 flask at approximately 1:6 dilution ratio meaning your seeding density in the new T25 cm^2 flask would be 0.25 million which is 4 times less than what is recommended for PANC-1 cell line for a T25 cm^2 flask.
As you very well know that low seeding density in cell cultures can lead to cell death. When cells are seeded at a low density, they may not have enough cell-cell or cell-matrix interactions, which can trigger programmed cell death. Additionally, a lack of cell-cell communication at low densities can prevent the release of sufficient growth factors into the culture medium, further hindering cell growth and potentially leading to death.
So, seeding an appropriate number of cells in T-25 flask for optimum growth of PANC-1 cells is essential.
Regards,
Malcolm Nobre
Hello Malcolm Nobre ,
Thank you for the answer, I hope this will work.
I also noticed that when the seeding density is low, small dark particles appear in the spaces between the cells. These look like cellular debris or possible precipitates, but I'm not sure exactly what they are. Do these structures also form when PANC-1 seeded at low density?
Thanks in advance,
Regards
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