I am doing differentiation studies with MSC.
I was inducing osteo- and adipo- differentiation in the same 24 well plate, and I want to stop the osteogenic differentiation and fix it for assay. However, adipogenic differentiation still needs time (about a week).
Can I fix some of the wells and still culture the rest of the wells?
Thanks!
Hi Chi Woo Yoon,
Our lab regularly uses Osteogenic and Adipogenic differentiation of MSC.
We always culture in either 96 or 24 wells and always keep Oste and Adip assays separate.
I do believe that fixing the plates and placing back in the incubator may have a adverse effect on the fixed samples (possibly rolling up of the cell layer), as they are sitting at 37 degrees rather than room temp. Also you might get evaporation of residual fixative within you plates which could potentially be detrimental to the cells still living.
I would highly recommend you use separate plates.
Cheers Sarah
Hi Chi Woo Yoon,
Our lab regularly uses Osteogenic and Adipogenic differentiation of MSC.
We always culture in either 96 or 24 wells and always keep Oste and Adip assays separate.
I do believe that fixing the plates and placing back in the incubator may have a adverse effect on the fixed samples (possibly rolling up of the cell layer), as they are sitting at 37 degrees rather than room temp. Also you might get evaporation of residual fixative within you plates which could potentially be detrimental to the cells still living.
I would highly recommend you use separate plates.
Cheers Sarah
Hi Chi Woo Yoon,
We have done this without any problems in our lab however you will need to use an alcohol based fixative (we use 95% methanol for 10 minutes) rather than formaldehyde as we have found that formaldehyde will kill cells in adjacent wells.After fixation wash the fixed wells with PBS then add plenty of PBS and maintain the levels until you are ready to stain. Switch to formaldehyde based fixation for the adipo if you are planning to stain with oil red o.
Tina
Do you know how long can cells be kept in PBS after fixation (formaldehyde) without any problems in alizarin red staining?
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