# Selector+
## 3 Results and Discussion
### Cell Analysis Post-Selection  
Viable cells were counted pre- and post-analysis using the exclusion dye erythrosine, and afterwards through flow cytometry analysis to detect expression of Annexin V, the apoptotic marker. Before Celector(r) analysis, HL60 cells showed a viability of 80%, whereas SW620 had a viability of 92% (Figure 3A). After the analysis and sorting procedure, collected cells, excluding the void, were counted and showed a higher viability--with most of the dead cells eliminated in the void, the first two minutes of the run. The viability of HL60 rose to 92%, and of SW620 from 92% to 99% (Figure 3A). Figure 2: Celector analysis for SW620 cells. Representative fractogram of SW620 cells at passage 1 in culture and collection time of the main population (**A**). The cell profile was monitored after in vitro culture for the CTRL cell population and for the collected cells, post-culture. After every enzymatic detachment, cells were analyzed for both conditions, and at p2, CTRL cells exit the channel earlier than POST cells (**B**), while at passage 7, both conditions showed similar elution times and fractogram profiles (**C**). The distribution of the percentage of cells during the analysis showed a difference in behavior during culture conditions, from a mostly homogenous situation to a higher presence of cells in F2 for the POST-derived cells (**D**). The cell diameter was measured, and CTRL cells showed a higher dimension at p2, and POST cells a stable dimension over the passages (**E**). When the population was decomposed into 2 fractions, F1 and F2, the resulting F1 cells are bigger than F2 cells at p2 and p7 (**F**). The representation of the average diameter as a function of time showed similar dimensions between the two conditions (**G**). Statistical analysis was performed using the 2 way ANOVA test and mean and standard deviation were graphed (******* and ******* \(p<0.0001\)**). In addition, to prove \(\text{C}\text{elector}^{\text{B}}\)'s ability to maintain cell viability, and to enrich the most vital cell population without cell damage, staining for the apoptotic marker Annexin V was performed and confirmed the exclusion dye counting data: the HL60 cell control population possesses 22% apoptotic cells, and the sorted cells only 5% (Figure 3B); SW620, as shown in the fractogram (Figure 3C), did not show an elevated void and the population was mostly alive with a viability of 92%. Annexin V staining showed that the percentage of early apoptotic cells was stable, around 2%, in both conditions. Moreover, to prove the maintenance of morphology and proliferative potential, images were captured via light microscope and immunofluorescence staining for specific markers were performed. SW620 cells showed a similar morphology using the bright-field microscope (Figure 4A) and by the expression of F-actin filaments to the stress cytoskeleton (Figure 4B). We could observe bigger colonies of cells, probably caused by the presence of more viable cells in the selected population compared to the control population due to the removal of the dead cells in the void. HL60 showed the same morphology, having the same ability to form small cell aggregates during culture conditions (Figure 4A). Figure 4: Representative images of HL60 and SW620 cells by light microscope (**A**) and expression of F-actin (**B**) in SW620 cells by immunofluorescence did not show any alteration in the cytoskeleton of  
Figure 3: Viability count (**A**), apoptotic cells positive for Annexin V staining (**B**,C**) in pre- and post-processed cells for both cell line HL60 and SW620, and a representative fractogram of the two. selected cells. The proliferative characteristics of cells were analyzed by immunofluorescence staining for Ki67 (**C**,**D**) and quantified by Image J post-processing analysis by measuring the percentage of the stained area (**E**). Statistical analysis was performed using the 2 way ANOVA test and mean and standard deviation were graphed (*** \(p\) < 0.001).