Probing cell metabolism using live-cell imaging and analysis
Cancer cells exhibit metabolic rewiring to support increased rates of proliferation and survival in the tumor microenvironment. This phenomenon is recognized as a hallmark of cancer. Recent studies also point to the importance of understanding the complex relationship between cancer cells and cancer-associated stromal cells, specifically evaluating how metabolic symbiosis promotes drug resistance. Researchers employ a wide-variety of biological techniques in order to expand their knowledge of cancer-associated metabolic changes and for the development of anti-cancer therapies. However, evaluation of metabolic changes in complex cancer cell models as they proliferate and interact with their environment are limited by current techniques.
Traditional techniques for measuring metabolic changes often:
- Cannot distinguish cell-type-specific metabolic changes in complex co-culture models
- Use indirect, byproduct measurements of metabolism as opposed to directly measuring metabolic ATP output
- Do not integrate confirmation of cell morphology
- Analyze an endpoint, rather than perform kinetic evaluation
- Do not incorporate physiologically relevant environment conditions, thus introducing potential artifacts
The Incucyte® ATP Assay is an end-to-end solution consisting of instrumentation, software, and reagents that enables direct analysis of ATP facilitating the understanding of metabolic change in cancer cells. Evaluate metabolic changes like never before!
Watch the Incucyte® ATP Assay for Live-Cell Analysis video
Incucyte ATP Assay
It Is Now Possible to Distinguish Cell-Type-Specific, Long-Term Metabolic Changes in Advanced Cell Models With Direct ATP Measurements
Gain unprecedented access to cell-type specific metabolic changes in advanced models with continuous imaging and analysis. The Incucyte® ATP Assay allows for direct measurements of cytosolic ATP for the characterization of tumor cell models in physiologically relevant conditions. Distinguish cell-type specific changes in ATP with integrated qualitative confirmation of cell morphology in real time with the Incucyte® SX5 Live-Cell Analysis System configured with an SX5 Metabolism Optical Module, Incucyte® ATP Analysis Software Module, and Incucyte® CytoATP Lentivirus Reagent Kit.
- Novel, non-perturbing reagent - Express a patent-pending, genetically-encoded fluorescent ATP indicator using the Incucyte® CytoATP Lentivirus Reagent Kit - compatible with a variety of cell types
- Analyze cell-type specific ATP changes - Capture and analyze dynamic cytosolic ATP changes to distinguish cell-type specific effects in advanced cell models
- Conduct temporal studies of ATP - Generate real-time ATP measurements for greater biological insight into impact of treatments - no concatenated endpoints necessary
- Confirm changes in cell morphology - Inspect changes in cell morphology with HD phase images at every time point
Novel, Non-perturbing Reagent
Express Incucyte® CytoATP Lentivirus Reagent in a variety of cell types for direct measurements of cytosolic ATP in live cells.
Figure 1. Efficient, non-perturbing labeling of living cells enables direct measurement of cytosolic ATP. HeLa cells were infected with Incucyte® CytoATP Lentivirus and subjected to puromycin selection to generate a stably expressing population. Morphology (A) and proliferation (B) of CytoATP-expressing cells were unchanged compared to parental cells. (C) HeLa cells stably expressing CytoATP seeded at 4,000 cells/well in a 96-well microplate were exposed to a combined treatment of 2-deoxy-D-glucose (2DG) and potassium cyanide (KCN). Quantification of CytoATP fluorescence images captured over 3 hours reveals a rapid, concentration-dependent depletion of ATP resulting from concurrent glycolysis and oxidative phosphorylation blockade.
Analyze cell-type specific ATP changes
Automatically measure and visualize cell-type-specific changes in cytosolic ATP for greater insight into the dynamic tumor environment.
Figure 2. Distinguish impacts on ATP in a specific cell type in monoculture or advanced cell models. The triple negative breast cancer (TNBC) cell line HCC1806 and receptor-positive breast cancer cell line MCF7 stably expressing CytoATP or CytoATP Non-binding Control were seeded in the presence or absence of CCD14086SK fibroblasts (8K cells/well). Identification of cellular changes in ATP (color scaled masking) is performed using the integrated Incucyte® ATP Analysis Software Module. Masked images (A) provide visualization ATP in HCC1806 cells treated with 1 µM CB-839 in monoculture and in co-culture with CCD14086SK cells. Kinetic data shows a more sustained depletion of ATP in TNBC lines (B) following CB-839 treatment compared to MCF-7 (receptor-positive cell line) grown in monoculture (C). However, co-culture with stromal cells mediated resistance to CB-839 in TNBC cells, while receptor-positive cell lines displayed no differential effect. IC50 curves depict 72 h (B) and 15 h (C) time point data.
Conduct Temporal Studies of ATP Dynamics
Generate real-time metabolic data for greater biological insight into both short-term and long-term ATP dynamics in response to treatments.
Figure 3. Discriminate mitotoxic vs cytotoxic compounds with nondestructive, repeated measurements of ATP. NIH 3T3 cells stably expressing CytoATP or Non-binding Control were grown in standard conditions (Glucose) or growth medium containing galactose substituted for glucose (Galactose) to drive dependence on oxidative phosphorylation for energy metabolism. Cells seeded at 12K cells/well in a 96-well microplate and fluorescent images (10X) were treated with nontoxic (Ambrisentan), cytotoxic (Chlorpromazine) or mitotoxic (Nefazodone) compounds. Kinetic data reveal a rapid depletion of ATP in cells treated with cytotoxic or mitotoxic compounds. While the response to cyotoxic compounds is similar in both media conditions, the leftward shift in IC50 of Nefazodone in cells grown in galactose is indicative of mitochondrial toxicity. A greater degree of separation is observed at early time points, demonstrating that kinetic CytoATP data can provide additional insight for evaluating compound profiles.
Confirm changes in cell morphology
Associate changes in cell morphology with ATP metabolism to reveal informative, temporal changes in cell behavior..
Figure 4. Enable qualitative inspection of cell morphology with HD phase images. HeLa cells stably expressing CytoATP or Non-binding Control seeded at 2K – 8K cells/well in 96-well microplates were treated with Chlorpromazine (60 µM), Etoposide (100 µM), or 2DG (20 mM) + KCN (2 mM), compounds that deplete ATP with variable time of effect highlighted by kinetic analysis (insets). HD phase images of cells (10x) acquired during depletion reveal drastic changes in morphology of cells treated with Chlorpromazine (21 h) and Etoposide (75 h), whereas cells treated with 2DG +KCN (60 min) show no significant morphological change compared to vehicle control.