In the first part of the LP, we performed excitation scans of Fura-2 in solution, with and

without Ca2+:

Figure 1: Excitation scans of Fura-2 in solution.

Figure 1 shows the change of the spectral properties of Fura-2 with increasing Ca2+
concentrations.
The interpretation of the results is as follows:
1) Fura-2 displays two different excitation maxima: one maximum around 380 nm (Ca-
free, black curve) and one maximum around 340 nm (Ca-bound, red curve).
2) When Ca2+ ions bind to Fura-2, the fluorescence at 380 nm excitation decreases and
the fluorescence at 340 nm excitation increases at the same time. These changes are
indicated in Figure 1 by the arrows with dashed lines.
3) The point at which all curves intersect is called the isosbestic point. At this point the
fluorescence of Fura-2 is constant and independent from Ca2+ ions.
 In the second part of the LP, we introduced Fura-2-AM into cells (mouse fibroblasts) and
applied ATP to induce a cellular Ca signal.
At the beginning of the recording the cell is at rest and [Ca2+]i is low. To determine the
change in [Ca2+]i, we illuminate the cell in an alternating fashion at 340 nm (to monitor Fura-2
at high Ca concentrations) and at 380 nm (to monitor Fura-2 at low Ca concentrations) and
collect the emitted fluorescence for both excitation modes separately. In our recordings we
switched between 340 nm and 380 nm every second.
Figure 2A shows the corresponding fluorescent traces: F1 in red (excitation at 340 nm)
and F2 in blue (excitation at 380 nm).

Figure 2: A ratiometric Fura-2 measurement of an ATP-induced Ca signal in a mouse fibroblast.

When the Ca signal develops and [Ca2+]i increases (grey box in Figure 2), then more
2+
Ca will bind to Fura-2. As a consequence, F1 increases and F2 decreases at the same time.
When the signal is terminated, both fluorescent traces return to their corresponding baselines.
Figure 2B shows the computed ratio F1/F2, which reflects the real change in [Ca2+]i over
time. The two main advantages of using the ratio are:
1) Elevations in Ca2+ are displayed as an increase in ratio: the more Ca2+, the higher the
ratio.
2) The ratio is independent from individual changes of F1 and F2, which are not related to
Ca2+, e.g., movement of the cells, bleaching of the dye etc.
 Then we tried to induce different cellular Ca responses using ATP. Figure 3 shows the
different Ca signals that we have obtained during both sessions of the LP. For a better
comparison, Figure 3 shows only the computed Fura-2 ratios.

Figure 3: Different ATP-induced Ca signals of mouse fibroblasts in the presence and absence
of extracellular Ca2+.

The Ca signal of a fibroblast in response to ATP is SOCE. It has two components, first
a release of Ca2+ from the ER (denoted as one in Figure 3) that is followed by a delayed entry
of Ca2+ from the extracellular space via ion channels (denoted as two in Figure 3).
In the first experimental situation we have applied ATP without extracellular Ca (Figure
3A). In this scenario, ATP will only cause the intracellular release of Ca2+ from the ER, whereas
the ion channel activity is prevented (left part of Figure 3A). Application of ATP resulted in a
quick and large elevation of [Ca2+]i, which declines over time and returns to baseline. This is
the isolated release of Ca2+ from the ER.
 In the second experimental condition (Figure 3B), we applied ATP in the presence of
2 mM extracellular Ca. We recorded a Ca signal that was slightly different: at the beginning of
the signal, the response to ATP was similar, but then the Ca signal was extended and longer
lasting. This is clearly visible if you compare the arrows in Figures 3A and 3B, which indicate
the time points of signal termination. This prolongation of the Ca signal is an influx of Ca2+ due
to the activity of store-operated Ca2+ ion channels. The whole signal is the native Ca signal of
fibroblasts, which is called SOCE.
Some cells respond to ATP in a less stable manner and display frequent fluctuations
of the Ca signal (Figure 3C). These types of signals are called Ca oscillations and can be
found in many cell types. They are often observed in the presence of extracellular Ca2+.
However, oscillations can be caused by both, fluctuating Ca release from the ER or oscillating
activity of the ion channel. Without further experiments we cannot determine which one causes
the observed oscillations.