The TetonTM and Teton AtlasTM cytoprofiling assays allow on board segmentation using three Cell Paint markers: cell membrane, nucleus and actin. To achieve accurate segmentation, the on-instrument software must be able to identify clear signals coming from these three features. If there is sub-optimal intensity of the cell paint markers for cell membrane and nucleus, which are applied off-instrument, these can be titrated to achieve the best results.
Optimization of these features can be performed using our on-instrument optimization kits, Teton Custom Antibody Screening Kit or the Teton Onboard Cell Paint Imaging Kit, or our off-instrument Teton Cell Paint Probe Kit. With these kits, the pre-mixed cell membrane and nuclear probes are incubated for 5 minutes. To optimize intensities, we recommend following the Teton Optimization & Screening Guide varying the amount of time the probes are incubated using the following setup as an example. If intensity is too high with a 5 minute incubation time, times may be adjusted to explore shorter time points beyond what is shown below.
Example optimization time course in a 12-well slide kit | ||
1 | 2 | |
A | 2 minutes | 30 minutes |
B | 5 minutes | 20 minutes |
C | 10 minutes | 15 minutes |
D | 15 minutes | 10 minutes |
E | 20 minutes | 5 minutes |
F | 30 minutes | 2 minutes |
If you are completing an off-instrument optimization, visualize both the cell membrane and nuclear signal. Look for features with sufficient signal and good edges of the cells from the membrane signal
If you are completing an on-instrument optimization, once the run has completed, visually inspect within CytoCanvasTM. Look for features with sufficient signal and that the segmentation model has been properly applied to your samples. To do this, run Cells2Stats using the --visualization-only argument to first generate your visualization files. Then, review the visualization files in CytoCanvas. Visually inspect that you have clearly drawn boundaries around the nucleus and cellular membranes.