good morning,
I have a problem of repetibility for my IC50 on spheroid.
Indeed I am trying to determine the IC50 on a 3D culture in spheroid. I am doing a range of 10 concentrations in triplicate. In order to read the viability I use the kit "cell titer glow 3D" from promega following the following protocol:
- Remove 100µl of media
- add 100µL of cell titer glow 3D
- incubation 20 min at RT under gentle agitation in the dark
-Incubation 20 min at RT without shaking (fluorescence stabilization).
-Finally reading the bioluminescence.
My problem is that I observe important variability between replicates and from one manipulation to another with the same cells and the same condition... however the manipulation is correctly done and my protocol is adapted from the commercial notice.
I wonder if I am the only one to observe a significant variability on 3d cultures for this type of experiment?
I ask myself if in view of the recommended incubation times it would not be better to dissociate the spheroid in a dissociation buffer before ( like for FACS reading) and then to lyse the cells with the cell titer glow in order to avoid an incomplete lysis
Thank you in advance for your advice!
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