Membrane Stability in Cell Culture

Last week I ran cell culture growth experiments on RTPed (800C 5 minutes in Ar) samples from W567 (single slit 100um x 1000um). For this study I tracked discoloration w/cells, membrane stability w/cells, and the bead assay w/cells to see if we can predict if a membrane used in cell culture is intact by monitoring discoloration of a separate sample. A detailed protocol can be found here. It should be noted that different samples were used for all conditions. The results are shown below.

From this study it is clear that membrane failure occurs during the early stages of discoloration. Additionally, membrane failure occurred between 1 and 2 days of culture, which is earlier than we would like. It should be noted that discoloration of samples used in the cell growth images (not shown) occurred earlier than discoloration of the discoloration test samples. This may be due to the cloning rings used in the cell growth samples, which may isolate cellular waste, accelerating discoloration.

Moving forward I plan on running two more experiments:

1. Variation of RTP conditions to improve membrane lifetime in culture. The goal of this experiment is to work towards an optimized RTP condition.

2. Variation of cell density on RTPed and non-RTPed samples. The goal of this experiment is to see if there is a difference in membrane stability depending on how many cells are present, with the use of non-RTPed samples as controls.