I'm doing OGD experiment for mature oligodendrocytes cell lines and it's not showing an expected amount of cell death and apoptosis (damage was not significant compared to the control). I’ve exchanged the culture medium for pre-warmed OGD medium. After that I transfer the OGD plates into the hypoxia chamber which flushed through the N2 (95 %)/CO2 (5 %) gas mixture for 5 min with an open outlet valve to achieve an N2 -enriched atmosphere. Following that, I clamp the inlet and outlet valves and bring back the hypoxia chamber to the incubator at 37°C for 2 or 2.5 or 3 hours (I’ve already tried different time point of incubation).
After OGD exposure, I remove the plate from the hypoxia chamber; replace OGD medium with DMEM containing glucose in addition to MTT solution and incubate it for 2 hours (reperfusion).
After 2 hours, I discard the old medium, added DMSO and measured the cell viability.
Do you have any suggestion to get the desired damage for my cells?
or is there any other model rather than OGD facilitate damage of my target cells?
After the OGD, try leaving your cells in culture for 24hours before doing the MTT assay. In our cells, the damage is initiated but the mitochondria are still functional immediatly after OGD which would not reflect cell damage or death with an MTT assay.
After OGD, place your cells in your DMEM media. Wait for18-24 hours before adding the MTT solution and then analyse with the DMSO.
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