Hello there,

I am trying to analyze the changes in the intracellular calcium concentration in the isolated synaptosomes and gliosomes after subsequent purinergic receptors stimulation. Based on the literature we know that such stimulation evokes intracellular calcium influx.

Briefly, after the isolation of synaptosomes/gliosomes, they were loaded with 5 μM Fluo-4 AM for 45 min at 4 °C (we tried also 37 °C) in a standard HBSS. Afterward, the cells were washed once (or twice) with HBSS and the fluorescence was measured using a microplate reader set at 485 nm excitation and 538 nm emission wavelengths. After determining the baseline fluorescence of the cells, the changes in fluorescence after the addition of the test compounds were recorded every 20 s for 8 min.

My problem is that after treating the synaptosomes/gliosomes the signal is the opposite of expected. After purinergic receptors stimulation, the intracellular calcium concentration decreases.

We thought that maybe cells were not loaded with Fluo-4 but when compared to control blank samples we see the difference. We supposed that the problem could be with methodology.

Have any of you experienced a similar situation? I will be grateful for every hint!