Figure 1A. Track changes in cellular parameters associated with T cell activation. Subset classification based on cell enlargement and morphological change in activated T-cells.  PBMCs were treated with anti-CD3 and IL-2, or vehicle control, and monitored over time with the Incucyte^® Live-Cell Analysis System.  (A-D)  Activation induces a time-dependent increase in average cell area and eccentricity (all cells).  Note the change in eccentricity precedes the increase in area.  The cell-by-cell area distribution (E) and density plots (F-H) highlight the increased heterogeneity over time following activation, and the appearance of a population of large cells with high eccentricity. Values shown are the mean ± SEM of 12 wells.

Figure 1B. Analyze subsets of activated T-cells: CD71 upregulation. (A-C) Cell-by-cell density plots shows increase in CD71 (FabFluor-488 labeled) fluorescence as a function of time and cell area (phase) in activated T-cells.  (D) Dynamic changes in the proportion of CD71+, but not CD4+ cells, follows activation. (E) Subset analysis shows preferential upregulation of CD71 expression in large (>100 mm²) vs small (<110 mm²⁾ cells. These data demonstrate an activation-induced, time-dependent increase in CD71 expression and a strong linkage between cell size and CD71, with >90% of large cells expressing CD71 after 4 days of activation.

Figure 2a. Immunophenotyping validation via cellular identification and classification on the Incucyte^® Live-Cell Analysis System. Freshly isolated PBMCs from three donors were characterized for six CD markers and IgG control using either flow cytometry or live-cell Immunocytochemistry analysis using FabFluor-488 labeled Ab.  A strong correlation was observed between two methods when considering the mean values from the three donors (A), or each donor alone (B). 

Figure 2b. Phenotyping HER2 expression in various cell types using cell by cell analysis on the Incucyte^® Live-Cell Analysis System. A549, SKOV-3 or SkBr3 cells were characterised for HER2 expression using FabFluor-488 labeled Abs. Images (top row) and quantification of fluorescence (bottom row) demonstrated lack of HER2 expression in A549 cells, heterogeneous expression in SKOV-3 cells and homogenous, high expression in SkBr3 cells. Isotype control responses represent the negative population.

Figure 3a. Generate robust cell cycle phase data suitable for pharmacological studies. Cell cycle phase distribution in HT1080 fibrosarcoma cells expressing the Incucyte Cell Cycle Red/Green Lentivirus Reagent (INSERT LINK) was quantified following cisplatin or fluorouracil (5FU) treatment. Response at 24h is shown in cell images (outlines show individual cells as identified by Incucyte Cell-by-Cell Analysis software) (A). Post-cisplatin treatment, percent of cells in the G1 phase (green fluorescence) decreases (B) and S/G2/M phase (red fluorescence) increases (C) in a concentration- and time-dependent manner, in line with its known mechanism of action to interfere with mitosis. After 5FU treatment, the percent of cells in the G1 phase increases (E) and in S/G2/M phase decreases (F) in line with 5FU’s known effect on DNA synthesis. The concentration response curves (D&G) show the compound effects at 24 h on all detectable phases of the cell cycle.

Figure 3b. Track Cell Health in sub populations of cells: Time course of HT1080 fibrosarcoma apoptosis following Camptothecin (CPT, cytotoxic) or cyclohexamide (CHX, cytostatic) treatment. Cell health was determined with multiplexed readouts of Incucyte^® NucLight Red (nuclear viability marker) and non-perturbing Incucyte^® Caspase 3/7 Green Reagent (apoptotic indicator). Cell images show response at 24 h with associated classification masking (A & E). Cell subsets were classified based on red and green fluorescence using Incucyte Cell-by-Cell Analysis Software tools (B & F). After CPT treatment, there was a decrease in the red population indicating loss of viable cells, increasing red and green fluorescence indicating early apoptosis, as well as increasing green fluorescence indicating late apoptosis (C). After CHX treatment there was a lack of apoptosis (G). Concentration response time courses of the early apoptotic population are shown (percentage of total cells exhibiting red and green fluorescence, D & H). Values shown are the mean ± SEM of 3 wells.