Fig. 3

Effect of MSC-conditioned medium containing high and low levels of BDNF on cortical neuron cultures exposed to OGD. A The diagram shows the protocol used. Cultures were exposed at 7 DIVs to 3 h of OGD and 24 h of reoxygenation. Cells were cultured in standard medium (ctrl), mesenchymal stem cell medium (MSC med), or conditioned medium obtained from donor 5898 (low BDNF) or COMM2 (high BDNF) samples during the reoxygenation phase for 24 h. At 8 DIVs cells were processed for immunocytochemistry and cell-based HCS analysis. B, C Graphs show the percentage of condensed nuclei measured in pure neuronal (B) or mixed astrocyte/neuron cultures (C) exposed to normoxia control conditions or OGD. D Representative images of pure neuron and mixed astrocyte/neuron cultures exposed to normoxia under control conditions, or to OGD under control conditions, with the culture medium of mesenchymal stem cells (MSC med), or with the conditioned media. The following stainings were used: beta-III-tubulin (green; neurons), GFAP (red; astrocytes) Hoechst (blue; nuclei). Images were modified increasing brightness for visualization purposes, but High Content Screening software process the raw images independently from post-acquisition modifications. The same brightness/contrast manipulation was applied to the whole image set. Scale bar: 200 µm. Statistical analysis. Columns represent the mean + SEM. One-way ANOVA followed by Tukey’s post-test. Asterisks represent the differences between the indicated groups (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)