Vala Sciences

We recommend seeding cardiac myocytes at 30,000 to 40,000 cells/well in 96-well dishes, and maintaining the cells at least overnight prior to screening (however, cells derived from adult rodent hearts can be seeded at a lower density, and utilized the same day as isolation, if desired). Ideally, cells microtiter plates coated with Matrigel (BD Biosciences) and cultured to achieve the near-confluence density. To promote the cardiomyocyte differentiation and maturation, the culture medium should be replaced every 24 h starting the day 1 following seeding, with the medium containing DMEM, 2% FCS, and Pen-Strep.

To record transients of intracellular cardiomyocytes calcium concentration we recommend using Fluo-4 family of calcium indicators (Life Technologies). Fluo-4 AM is an organic fluorescent calcium indicator that is widely used for intra-cellular measurement of calcium signaling in optical microscopy, high-throughput screening and high-content screeningapplications. Its visible wavelength excitation, high sensitivity, and large fluorescence increase upon binding Ca2+ have made it the indicator of choice for many scientific applications.

Fluo-4 NW (“no wash”) fluorescent indicator is the latest development in fluorescent calcium indicators family. The fluo-4 NW assay achieves larger increases in fluorescence intensity than standard Fluo-3 and Fluo-4 assays, as export of fluo-4 from the cells is inhibited. The Fluo-4 NW indicator is relatively nonfluorescent and stable in pH 7–7.5 buffer for several hours. This feature allows to load Fluo-4 NW inside cells and obtain stable signal during recordings for approximately 60 min.

We recommend preparing reagents on the day that you will perform your experiments. To record the real-time changes in intracellular calcium levels using the KIC, cardiomyocytes are labeled with the nuclear label Hoechst-3342 (400 ng/ml) and with Fluo-4 NW (3 µg/ ml) simultaneously. Cells should be co-labeled with Hoechst-3342/Fluo-4 NW mixture for 20 min at 37°C. Than cells should be incubated with Fluo-4 NW for additional 15 minutes at 37°C, and at room temperature for an additional 25 minutes. We have found that the best results are obtained with this combination of times and temperatures for incubation and measurement.

After labeling, the cells should be washed twice and transferred into appropriate recording solution. For fluorescence measurements we recommend using instrument settings appropriate for excitation at 494 nm and emission at 516 nm. A 488/520 nm excitation/emission filter set works well for Fluo-4 recordings.

The IC 200 KIC module can be used with standard 86 mm x 128 mm multi-well plates. The IC200 software enables user definable well formats to fit within the standard plate footprint.

Standards are set by the Microplate Standards Development Committee of the Society for Biomolecular Screening.

KIC Guidelines for a microtiter plates selection:
1.    All plates should have a bottom thickness consistent with #1 or #1.5 coverglass (170 μm) for use with the IC 200 System standard objectives.
2.    All plates must have flat clear bottom.
3.    Glass or optical quality plastic plates are recommended. The recommendation is stronger for higher resolution measurements and dimmer fluorescence.
4.    Black walls are recommended.
5.    Use sterile, tissue culture treated plates as needed for specific applications.

ATTENTION: Vala Sciences neither recommends the use of certain well plate type in preference to another nor guarantees the acceptability of the well plate to produce quality results. If you need information on a well plate not listed here, contact your Vala Sciences representative.

1.    Transfer the 96-well microtiter plate to the IC200 screening system.
2.    Place the plate onto the plate holder. Make sure that the plate is positioned correctly on the screening stage. Gently push on the plate rim until the plate skirt clicks into place.
3.    Move the electrode arm into working position. Secure the arm position by tightening the Fix Screw.
4.    Close the IC200 loading door.

Your system is ready for screening.

The IC 200 KIC allows the customer to deliver an electrical stimulation of cardiomyocytes at any time prior or during image capture. The stimulation protocol is synchronized with TIFF image sequence for further data analysis.

For data analysis, the IC200-KIC utilizes CyteSeer automated data analysis software. Please refer to CyteSeer Time-lapse Data analysis section for details.

For recording, a 96-well microtitier plate was transferred to an IC200 robotic plate positioner stage. The positioner stage moved the test wells into landing pad one at-a-time according to an experimental protocol. As a next step, IC200 performed auto-focusing using Nuclear labeling of cardiomyocytes as a target (Blue Channel). Simultaneously two platinum KIC stimulation electrodes were immersed into a test well.

Our experimental recording protocol included following steps:
First, EM CCD camera collected a single image of cardiomyocytes nuclei labeled with Hoesht.

An EM CCD camera then records a Fluo-4 signal reflecting dynamic changes in intracellular calcium concentration. Images were recorded as a series if TIFF images with a 30 Hz frame-rate. To collect the signal, excitation/emission parameters were set at 488/520 nm.

During the calcium signal recordings, electrical stimulation pulses were delivered to test cells through the KIC electrode pair.

After the completion of the recording sequence, electrodes were raised from the well to the initial ready position.

This series of steps is repeated over the entire set of selected wells to be studied.