2.7. Calcium imaging

Single cell calcium imaging, a method correlating the functional data based on calcium shifts operated by different intracellular and extracellular mechanisms integrated with their cell phenotypes, is a widely recognized mean to assess the response of neuronal and glial cells to local stimuli and has been used extensively in vitro (isolated cells), ex vivo (brain or spinal cord slices) and in vivo (two-photon microscopy) [40]. The method involves the use of a cell-permeable calcium indicator or genetically encoded calcium indicator that, once entered the cytosol or synthesized by the cell, responds to variations of the intracellular calcium concentration by changing the intensity of fluorescent emission or the ratio of fluorescent emission at two different wavelengths [41]. Here we used the cell permeant indicator Oregon Green™ that was loaded into SCSC as previously described [42]. Briefly, following treatment with MSC CM or control medium, SCSC were incubated with 250 μl loading solution for 1 h at 34 °C/5% CO₂. The loading solution consisted of 1 μl Oregon Green™/DMSO mix (50 μg Oregon Green™ 488 BAPTA ((1,2-bis(o-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid)-1AM in 4 μl DMSO) and 2 μl 20% pluronic F-127 acid (in DMSO)) in 1 ml Neurobasal medium. Slices were washed and mounted onto a confocal microscope (Leica TCS SP5) stage equipped with a buffer transfer system continually perfusing the preparation with oxygenated artificial cerebral spinal fluid (125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO₃, 1 mM NaHPO₄, 25 mM glucose, 1 mM MgCl₂, 2 mM CaCl₂ in dH₂O). Digitised time-lapse images were collected under 40x water immersion objective, (excitation wavelength 488 nm, emission wavelength 495–530 nm) at 204 ms frame intervals totalling 1000 frames. Gain, offset and pinhole were constant throughout imaging. SCSC activity was unchanged when challenged with 60 mM KCl, making it difficult to unequivocally identify calcium oscillating cells as neurons [43]; therefore, the term ‘active cells’ has been used thereafter.