There are a number of reasons for 'live' cells being detected in the bottom. Some reasons as to why this happens can include:
- Live cells 'slipping' into the bottom during harvest. This is more common when using a higher split line. With a higher split line, there is an increased likelyhood of cells from the top ending in the bottom. On the positive side, there is less chance of dead/ dying going into the top cells, so stringency can be paid off when we want to eliminate the possibility of dead and dying. However - yield can be affected, so a higher split line can be recommended only when yield losses are acceptable.
- Cells that are dying but not yet fully staining as dead can show up in the bottom. Ergo they have a permeated membrane, and have the characteristics of a dead cell during levitation, but not quite yet on the cell counter / stains
- Live cells stuck to debris / ECM /clumps that have fallen to the bottom. During the separation procedure, live cells that were not dissociated fully and released from their surrounding structure can end up in the bottom half of the channel. These cells will stain as live, alongside their neighbouring dead cells and debris. This would lead cellular counters to count the bottom half.
- Live cells have leaky membranes due to pre-processing steps. In some cases, it may be that processes's that the cells have undergone have left some of the live cells with permeable membranes. For exapmle
What should I do if I have high viability in the bottom channel?
If you find high viability in the bottom channel, there can be a few things worth investigating:
- Verify cell viability using a different counting method. Sometimes cell counters can provide unreliable estimates to the viability of the cells. This is more common with tissue dissociate samples, and when using single stain counters. Verifying a certain count using a separate counting method can corroborate the first count.
- Image the samples under a microscope. Imaging the output samples often can show that although the viability of the bottom (waste) output is high, there are many more clumps and debris in this sample. Therefore though viability is detected, it is not an optimal sample for downstream applications.
- Compare the counted cells with the Fractionation Analysis output. The Fractionation Analysis output reveals how much of the biomass was above and below the split line at harvest. In general, what is above the split line can be considered as viable cells. what is below can be considered dead cells and debris. Using the percentages above and below, along with the starting input number we can get an estimate of how many cells are viable in our output sample. Use this to corroborate or disprove the counted metric post levitation.