Methods for a Representative Kinetic Image Cytometer (KIC) Experiment
This is a representative methods section for a paper using the IC200 Kinetic Image Cytometer (KIC) system.
Human induced pluripotent stem (iPS) cell-derived cardiomyocytes (e.g., iCell™ Cardiomyocytes from Cellular Dynamics Int.) were utilized in our experiments. These cells represent a mixture of electrically active atrial, nodal, and ventricular-like cardiomyocytes that exhibit electrophysiological and biochemical responses typical for mature (non stem cell-derived) cardiomyocytes.
The cells were plated in 96-well 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 was replaced every 24 h starting the day X with the medium containing DMEM, 2% FCS, Pen-Strep 0.X mmol/L. Note that utilizing low concentrations of serum in the culture medium promotes that development of the calcium regulating infrastructure in cardiomyocytes via modulation of the key cardiac transcription factors (ref) and ultimately promotes development of contractile activity. Spontaneous contractions that serve as indicators of maturation of cardiomyocytes have appeared in our cell culture between days X and Y after plating, and at this experimental stage were observed using the bright-field microscopy.
To record the real-time changes in intracellular calcium levels in a High Content mode, cardiomyocytes were simultaneously co-labeled with the nuclear label Hoechst-33342 (XX µM) and with the fluorescent calcium indicator Fluo-4. In the majority of our experiments, we used the Fluo-4 NW kit from Life Technologies, and performed the loading according to the published labeling protocol. After labeling, the cells were washed twice and transferred into Tyrode’s solution supplemented with 10 mM glucose.
Electrostimulation and Data Recording
At the start of experiments, a 96-well microtiter plate with cardiomyocytes was transferred to the IC200 robotic plate positioner stage. The stage then moved the specific test well into the recoding position according to a user-specified experimental protocol (see the IC200 manual for details). During the next step, the IC200 autofocused on the sample, using the Hoechst-3342 labeling of cardiomyocytes’ nuclei as a target (Blue Channel, Excitation/emission 350 nm/465 nm). Such image-based autofocus has a distinct advantage of finding an optimal focal plane for each individual recording position in the well.
In parallel with the auto-focusing process, KIC field stimulation electrodes were immersed into the test well and positioned 100 µm above the bottom of the well. The frequency of electrical stimulation can be adjusted from 0.1 to 100 Hz using the CytoShop image acquisition software. Amplitude and duration of the electrical pulse was controlled by a stimulator XXX that allows varying these parameters in the range 1-100 V and 0.1-1000 ms, respectively. In our experiments, cardiomyocytes were stimulated at frequencies 0.5 - 4 Hz. To trigger cardiomyocyte contractions in a stable and reproducible manner, 5-ms monophasic pulses of 15 V were used. In fact, very short (~5ms) electrical pulses of low amplitude (15 V) are can sometimes initiate depolarizations in at least in a subset of the test cells, depending upon the state of maturity of the cardiac myocytes in the culture. Given that the passive membrane properties of cells can be effectively modeled as a simple RC circuit, it is not surprising that only a portion of the cells are firing as a result of any given electrical stimulation protocol.
When action potentials in the cardiomyocytes are triggered, the intracellular Ca concentration changes dramatically from 100 nM to 200 – 400 µM. Intracellular fluorescent calcium indicators will report these concentration changes as dynamic variations the fluorescence emission. Depending on the experimental goal, fluorescent signals can be recorded from cardiomyocytes that are either spontaneously active in the absence of any electrical stimulation, or paced using one of many custom stimulation protocols. In our experiments, the effects of different experimental conditions, including effects of drugs, on the calcium component of cardiac action potentials were monitored by recording 10 - 20-sec time-lapse movies at the 30 Hz frame rate utilizing Fluo-4 (Green Channel, excitation/emission 488/520 nm). After completion of the data acquisition/stimulation protocol, the electrodes are raised from the well, and the instrument repositions the plate to the next well to be analyzed.
All of the steps involving in the data acquisition are accomplished in an automated fashion, and are repeated for each well on the plate that has been specified for analysis by the user.