Acquisition of an IncuCyte S3 Live-Cell Analysis System

LEO Foundation Skin Immunology Research Center currently consists of four research programs/groups: 1) Skin Inflammation and Cancer, 2) T Cell Biology and Skin Inflammation, 3) Translational Skin immunology, and 4) Molecular immunology and Inflammation. Prof. Mads Gyrd-Hansen heads the Molecular immunology and Inflammation group, whose aim is to characterize and understand the molecular processes that control immune responses, with a particular focus on innate immune responses. Through this, the group aims to advance our understanding of the molecular aetiology of inflammatory skin diseases and other immune disorders, which ultimately may pave the way for improved treatment strategies.

The IncuCyte Live-Cell Analysis System is the first real-time system that automatically captures and analyzes images of living cells around-the-clock for days, weeks, or months, while cells remain undisturbed inside a standard tissue culture incubator. The IncuCyte system is intended to be used for studying cell viability, migration, chemotaxis, invasion, activation, etc.

Sartorius Lab Instruments is the sole distributor in Denmark of Incucyte Live-Cell Analysis Systems manufactured by Essen BioScience Ltd. The IncuCyte S3 Live-Cell Analysis System is the only system available on the market with the following capabilities:

1. The automated image acquisition and analysis system can operate and capture images from within a standard tissue culture incubator so that precise control of temperature, humidity and other

environmental factors such as CO2 and oxygen can be maintained. The system is fully automated, allowing for hands-free operation for periods exceeding 25 days and is designed to autofocus and auto expose without intervention during this time period. The automated imaging system can return to the same location in a repeated fashion without error over this same time period. These features enable us to record cell behaviour (e.g. differentiation, cell death, activation) over time without unintended disturbance of the experiment.

2. The optics move to the areas being imaged. The cell culture vessels therefore remains stationary during this process, which is critical for our experiments. Stationary optics and stage driven vessel movement are not acceptable.

3. The system is capable of simultaneously imaging and analyzing a mixture of 6 assay plates that conform to the ANSI/SLAS standard for assay plates. These include 384-well microplates, 96-well microplates, 48-well plates, 24-well plates, 12-well plates, and 6-well plates. Each assay plate is able to run a different application, accommodating six different assay applications in parallel. It can accommodate a wide-range of standard plastic tissue culture vessels, chambered slides and microslides. This gives critical flexibility in our experimental designs.

4. The system has the following objectives on an automated turret: 4x PLAN, 10x PLAN FLUOR, and 20x PLAN FLUOR. This enables high definition phase contrast imaging and fluorescence imaging at two fluorescent wavelengths (red: ex565-605nm, em625-705nm; green: ex440-480nm, em504-544nm). The fluorescence optics are capable of reading YoPro-3, mKate2, GFP, YFP, Alexa 488, intercalating DNA dyes, fluorescein or fluorescein derivatives. This enables monitoring of multiple cellular parameters, which is important for our experiments (e.g. monitoring cell death in co-culture experiments with fluorescently-labelled cells).

5. The software is able to mask, quantify and generate time based curves based on fluorescence metrics including but not limited to: Fluorescent Count, Fluorescent Average Area, Fluorescent Total Area, Fluorescent Confluence, Fluorescent Mean Intensity, Fluorescent Average Integrated

Intensity, Fluorescent Total Integrated Intensity, and Fluorescent Eccentricity. This allows lab members to obtain reliable and objective data without the need for extensive training.

6. Control of the system is distributed over a network and the client software is able to

elicit control of the automated image acquisition and analysis system from any networked computer. Unlimited licensees of the client software is available, which allows users to operate the instrument and analyze experiments without using a client computer license key or dongle. This is important for efficient use of the instrument by multiple lab members.