TABLE 5.
Perkin Elmer Columbus System parameters for script analysis (astrocyte cultures).
| Cell feature | Columbus building block | Specifics | Method | Output name |
| Nuclei | Find Nuclei | Channel: Hoechst 33342 | Method: B Diameter: > 30 μm Common Threshold: 0.4 | Population: Nuclei |
| Calculate Intensity Properties | Channel: Hoechst 33342 Population:Nuclei Region: Nucleus | Method: Standard, mean | Properties: Intensity nucleus Hoechst 33342 | |
| Calculate Morphology Properties | Population:Nuclei Region: Nucleus | Method: Standard, area, roundness | Properties: Nucleus Morphology | |
| Select Population | Population: Nuclei | Method: Filter by property Nucleus area (μm2): > 40 Nucleus area (μm2): < 1000 Intensity nucleus Hoechst 33342 mean: < 18512 Nucleus roundness: > 0.4 | Population: Viable nuclei | |
| Vimentin + cells | Find Cytoplasm | Channel: Alexa 488 Nuclei: Viable Nuclei | Method:A Individual threshold: 0.15 | |
| GFAP + cells | Find Cytoplasm | Channel: Alexa 633 Nuclei: Viable Nuclei | Method:A Individual threshold: 0.15 | |
| RAN + cells | Calculate Intensity Properties | Channel: Alexa 488 Population: Viable nuclei Region: Nucleus | Method: Standard, mean | Properties: RAN intensity in nucleus area (Alexa 488) |
| Calculate Intensity properties | Channel: Alexa 488 Population: Viable nuclei Region: Cytoplasm | Method: Standard, mean | Properties: RAN intensity in cytoplasmic area (Alexa 488) | |
| RANGAP1 + cells | Calculate Intensity Properties | Channel: Alexa 488 Population: Viable nuclei Region: Nucleus | Method: Standard, mean | Properties: RANGAP1 intensity in nucleus area (Alexa 488) |
| Calculate Intensity properties | Channel: Alexa 488 Population: Viable nuclei Region: Cytoplasm | Method: Standard, mean | Properties: RANGAP1 intensity in cytoplasmic area (Alexa 488) | |
| lamin-B1 + cells | Calculate Intensity Properties | Channel: Alexa 594 Population: Viable nuclei Region: Nucleus | Method: Standard, mean | Properties: lamin-B1 intensity in nucleus area (Alexa 594) |
| Calculate Intensity properties | Channel: Alexa 594 Population: Viable nuclei Region: Cytoplasm | Method: Standard, mean | Properties: lamin-B1 intensity in cytoplasmic area (Alexa 594) |