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Tumor cells were cultured in 12-well plates in the presence or absence of CBD and/or cannabinoid antagonists for 24 h as described above. The percentage of apoptotic cells on the total cell population (adhering + detached cells) was evaluated as previously described ( Ceruti et al. 2000 ). Briefly cells were collected washed and centrifuged at 200g.
The different potency of CBD versus THC was also confirmed in our how to make hash oil for brownies experimental protocol where THC affected the viability of our cell lines after 4 days of exposure and with an IC50 of about 3 to 4 ?M (data not shown). In the present study we showed for the first time the ability of CBD to induce programmed cell death in human glioma cells through two independent methods based on the appearance of a hypodiploid DNA peak after PI staining of nuclei and detection of apoptotic DNA with monoclonal antibody to ssDNA. These results demonstrated that CBD was capable of inducing cell death and that this mechanism correlated with the reported inhibition of cell growth found in both in vitro and in vivo studies. These results are in accordance with previous reports demonstrating cannabinoids as agents able to cause apoptosis in both in vitro and in vivo experiments ( Guzman et al. 2002 ).