Combined Live Cell and Flow Cytometry Analysis of Immune Cell Killing
In vitro cell biology models are becoming ever more complex to better mimic in vivo studies. However deciphering the meaning of results gained from the complex biological interactions becomes more difficult and requires a greater understanding. Combining specialist technologies provides the user with a more detailed analysis in order to delve deeper into the biology and make more physiologically relevant conclusions. In order to do this, workflows must be developed linking technology platforms using a streamlined approach that is simple to follow as well as using both time and samples efficiently.
Incucyte-iQue Workflow Concept
The Incucyte-iQue workflow uses the immune cell killing application to describe how a single assay plate can be used to acquire real-time interaction, daily cytokine concentration and T-cell activation profiles.
- Adherent or non-adherent target cells expressing Incucyte NucLight Green are co-cultured with immune cells (label free).
- Tumor cell proliferation and death are monitored using the Incucyte. Utilising the fluorescent NucGreen count of the target cells and the addition of Annexin V to the culture.
- Daily cytokine concentration can be monitored throughout the experiment by sampling from the assay plate and using the Qbeads (capture beads and detection antibodies).
- Incucyte automated image analysis enables selective quantitation of tumor cell death (basic analyzer) as well as PBMC proliferation and shape change (Cell-by-Cell).
- Cells are lifted (following the Incucyte-iQue Workflow protocol) and labelled using the TCA kit.
- T-cell subset and activation profiles are then quantified using a pre-defined gating strategy.
Kinetic cytokine measurements using non-perturbing low volume supernatant sampling maximising sample usage with minimal manipulation
- 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.
Easy to use, mix-and-read assays using Intellicyt-Incucyte compatible reagents
- 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.
Combined subset analysis with spatial and morphological quantification
- 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.
Additional biological insight providing a greater depth of physiological understanding
- 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 72 h. Each data point represents mean ± SEM, n=3.
|IFNγ option 1||Qbeads Assay Builder|
|TNFα option 2|
|T Cell Activation Cell and Cytokine Profiling Kit|
|Platform||Compatible with iQue3 VBR configuration|
|Available Sizes||Catalog Numbers||Order Now|
|1 x 96 well||90560||T Cell Activation Cell and Cytokine Profiling Kit|
|5 x 96 wells||90561|
|1 x 384 wells||90562
|5 x 384 wells||90563|
|NucLight Green Lentivirus Reagent|
|Available Sizes||Catalog Numbers||Order Now|
|0.2mL; 1x106 TU/mL||4475 or 4626||NucLight Green Lentivirus Reagent|