Incucyte^® - iQue^® Workflow

Monitor the full time course of tumor cell killing (>5 days), with non-perturbing supernatant sampling to determine cytokine concentrations.

Figure 1. Chronological events can be identified with the combination of technologies. 

Effect of CD3/CD28 Dynabead activation in a co-culture immune cell killing assay. SKOV-3 cells were plated at a density of 4,000 cells per well and were allowed to adhere for 4 hours. Serial dilutions of Dynabeads were then added to wells prior to the PBMCs, which were plated at 10,000 cells per well. Daily supernatant sampling (10 µL) was performed for cytokine analysis.

Temporal data demonstrates a timeline of events, whereby an initial increase in cytokine secretion (A, B) was detected which preceded an increase in effector cell proliferation (C) this then resulted in a decrease in target cell numbers (D). Data were collected over a 120-hour period. Each data point represents mean ±SEM, n=3.

• Data from a single assay plate can be collected and combined to provide deeper biological insight in co-culture models
• Workflow is amenable to both adherent and non-adherent target cells

Figure 2. Easy to follow workflow for the combination of Incucyte^® - iQue^® for an immune cell killing assay.

• Automatically phenotype T cell subpopulations and quantify cytokine secretion
• Observe and quantify phenotypic changes in target and effector populations in real-time

Figure 3a. Target cell analysis is comparable across platforms. 

(A) Phase-contrast/fluorescent image shows a co-culture of SKOV-3 Nuclight Green transfected ovarian cancer cells (target) and unlabelled PBMCs (effector).

(B) Incucyte^® software enables direct image-based detection and quantification of target cells (green objects, outlined in pink) enabling target cell number quantification.

(C) 2D density plot iQue^® analysis demonstrates how the green labelled target cells can be separated from the negative PBMC population.

(D) Target cell frequency.

Figure 3b. Automatic T cell subset analysis is achieved using a pre-determined gating strategy. 

User-friendly TCA-kit allows for the identification and quantification of T cell activation markers with minimal user intervention.

Deeper biological insight can be gained from the combination of technologies.

Figure 4. Quantify antibody-dependent cell-mediated cytotoxicity, changes in cell interaction and T cell activation in one assay. 

Effect of an anti-hCD3xCD19 BiTE antibody activation in a co-culture of non-adherent target cells and PBMCs. Ramos cells (green) were plated at 15,000 cells per well. BiTE antibody was added to wells prior to PBMCs addition (75,000 cells per well).

(A) The BiTE antibody induced a concentration-dependent killing of Ramos cells as measured by a loss in fluorescence intensity.

(B) Cell clustering was also increased in a concentration-dependent manner, a hallmark of immune cell killing. Differences in T cell activation markers were observed when BiTE stimulation was compared to Dynabead stimulation

(C, D) Morphological changes observed in the images

(E, F) provide qualitative verification. Data were collected over 72h. Each data point represents mean ± SEM, n=3.