I am trying to study osteocytes in vitro. Osteocytes are terminally differentiated in bone tissue however I am using a common cell line, MLO-Y4. This cell line has been transfected to be mitotic but I want to have an appropriate model of what really happens in vivo so I want to study these cells in a senescent state. Can I induce MLO-Y4 cells to become senescent using either drugs or osteocyte-specific culture conditions?
Roxanne Fournier,
Try adding retinoic acid and keep me posted please.
Best
Mannan
Try to add osteogenic differentiation medium: typically consist of: DMEM, 10%FBS, ascorbic acid (50ug/ml), Dexamethason (0.1 uM), beta-glycerphosphate (5mM) and keep cell in culture for three weeks, refresh medium every 3-4 days. This give you an indication of differentiation capabilities. Do a alizarin red or ALP staining after this. Best, Karoly
Hi Roxanne,
MLOY4 were derived from SV40TAg animals and, as such, they are transformed so it might be difficult to study senecnec in these cells. IDG(From Lynda) or Ocy454 (from my lab Spatz et al JBC2015) are 'conditionally" immortalized, meaning that you can turn off the SV40 Tag by changing culture conditions. I suggest switch to one of this alternative models. These cells will differentiate to mature osteocytes after being moved to "non-permissive" temperature (37-39) for 2-3 weeks (for Ocy454 11-14 days). We did studied senescence in these cells (Ocy454) after 3 weeks of culturing the cells at 37 C. cells were positive of SAb-gal.
Your cells may enter "senescence" in a modified manner like going to sleep without affecting viability. This probably can be accomplished by suspending the cells in the SCSR transport medium (available from NonInvasive Technologies LLC website is WWW.NonInvasive Technologies.com.
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