IncuCyte™ FLR

IncuCyte^FLR is the newest addition to the IncuCyte product line. This system supports the HD Imaging mode as well as a single fluorescent channel and two different resolution settings. This configuration is ideal for following live-cell reporters in applications which include stem cell research, co-culture angiogenesis, cell motility and proliferation.

In combination with the HD Optics, IncuCyte^FLR allows researchers to gather time-lapse images of a fluorescent channel along with high-contrast phase imagery. Prior to HD Imaging, the ability to overlay flourescence with phase was rarely utilized due to the poor quality of phase images in higher format microplates.

Like the rest of the IncuCyte^FLR is made to fit inside a cell culture incubator and it can accommodate most types of standard cell culture vessels including microplates, T-flasks and Petri dishes.

Image shown on the right side is an overlay of phase and fluorescent channels. MCF7 cells expressing nuclear H2B-GFP.

• Kinetic proliferation assays
• Kinetic, co-culture angiogenesis assays
• Monitoring live cell reporters over days
• Cell-cell interactions

Click here to see the full list of applications.

IncuCyte^FLR is ideal for following fluorescent reporters in live cells from within your incubator. As a demonstration of this utility, we have developed a kinetic proliferation assay using a nuclear GFP tag. One liability of traditional proliferation assays is that the drug effect is measured at a single time point (“end-point”). Using the non-perturbing GFP tag and the IncuCyte^FLR, proliferation assays can be run kinetically, thereby elucidating the effects of drug action on living cells over time. The IncuCyte software has the ability to discriminate and “count” objects. In the example presented here, “objects” are nuclei, thereby providing a direct, kinetic read-out of cell count, nuclear area, and other related metrics.

Population histograms of nuclear size (area) of MCF7 H2B-GFP cells treated with 1 mM hydroxyurea at a) time of treatment and b) 4 days after treatment showing the change in nuclear size distribution. Hydroxyurea inhibits cells growth by blocking cell cycle in S-phase.

a) at time of treatment

b) 4 days after treatment

MCF7 cells expressing nuclear H2B-GFP. Kinetic data showing nuclear count in response to various concentrations of hydroxyurea over 5 days. Data plot generated directly from IncuCyte software.

It is well-established that the metastatic potential of a tumor is in-part related to its ability to promote vascular angiogenesis (Folkman Nat Med Jan;191);27-31). As such, the multi-step process of angiogenesis has been a research focus area for anti-cancer drug development. In the last few years there have been new in-vitro models introduced, more advanced than the traditional Matrigel “tube-formation” assay and exhibiting more concordance with in-vivo models. One of these new models is a “co-culture” assay (Bishop, Angiogenesis Vol. 3: p.335-344 1999) which uses primary human umbilical vascular endothelial cells (HUVEC) in conjunction with dermal fibroblast cells. The formation of microtubes in this assay can take 10-14 days, ideally suited for the IncuCyte™ platform.

At Essen, we have adapted this assay for a kinetic read-out using IncuCyte^FLR. We accomplish this by labeling the HUVEC cell line with a non-perturbing GFP marker. We then use the IncuCyte to quantify the HUVEC cells, providing temporal metrics of tube area, tube length and branch points. This approach is very powerful in the determination of drug mechanism and allows for the specific effects of individual compounds or sequential compound dosing to be followed in real-time over the entire 10-14 day period.

Images of GFP-labled Huvec cells imbedded in dermal fibroblasts (co-culture) at Day 11 treated with a) vehicle control and b) 600 nM Angiopoietin I. Increased sprouting is consistent with cited reference. Koblizek Curr Biol. 1998 Apr 23;8(9): 529-532.

IncuCyte graphs of tube branching vs. time for increasing concentrations of Angiopoietin I. This protein growth factor promotes tube branching in a concentration and time-dependent manner (Click image to enlarge).

IncuCyte graphs of tube length vs. time for increasing concentrations of suramin. Response is both concentration and time-dependent (Click image to enlarge).             

Images of GFP-labled Huvec cells imbedded in dermal fibroblasts (co-culture) at Day 11 treated with a) vehicle control and...

b) ...1 mM Mycophenolic Acid. This compound exhibits strong inhibition of angiogenesis in vivo, but negative in traditional Matrigel tube formation assays.