Table 4.
CellInsight NXT Settings for Figures 3–5
| Channel 1: Nuclei | Value | |
|---|---|---|
| Dye/channel | Hoechst 33342 | Excite at 386 |
| Thresholding | Fixed intensity | 700 |
| Segmentation | Intensity | 300 |
| Object.Area.Ch1 | 245–2,400 | |
| Channel 2: Bacteria | ||
| Dye/channel | Alexa Fluor 647 | Excite at 650 |
| Thresholding | Fixed intensity | 5,000 |
| Segmentation | Intensity | 1 |
| ObjectCh2.VarIntensity.Ch2 | 675–4,000 | |
| Channel 3: Ubiquitina | ||
| Dye/channel | Alexa Fluor 555 | Excite at 549 |
| Thresholding | Fixed intensity | 4,200 |
| Segmentation | Intensity | 1 |
| ObjectCh3.VarIntensity.Ch3 | 180–2,700 | |
| Channel 3: LC3a | ||
| Dye/channel | GFP (stably expressed) | Excite at 488 |
| Thresholding | Fixed intensity | 1,000 |
| Segmentation | Intensity | 1 |
| ObjectCh3.VarIntensity.Ch3 | 180–2,700 | |
| ROI A Mask Creation | Channel 2 | |
| Target I | Channel 3 | |
a
Ubiquitin and GFP-LC3 are imaged in channel 3 in separate protocols.