I'm currently trying to induce and identify mineralization in my SaOS-2 cells.
The issue I'm having is that the whole well will stain red, and of course this intensity depends on how long the cells are left in the Alizarin Red S staining solution. I look at these cells over a time course of 0, 3, 6, 9 and 12 days. I grow these cells in DMEM F12 Glutamax media supplemented with 110mg/L sodium pyruvate and 1% penstrep. SaOS-2 cells were seeded at around passage 10 +/- a couple of passages.
My current culture protocol is as follows:
Seed SaOS-2 cells at 50,000 cells/cm2 in a 6-well plate and grow cells in DMEM F12 Glutamax supplemented with 110mg/L sodium pyruvate, 10% FCS and 1% Penstrep.
After the cells have reached confluence and I wash the day 0 in PBS and fix it with 4% PFA for 5 mins and store the cells at 4oC in 3ml PBS. and then add osteogenic media to the remaining wells.
Oestrogenic media = DMEM F12 glutamax supplemented with 110mg/L sodium pyruvate, 10% FCS, 1% penstrep, 50ug/ml ascorbic acid and 5mM Beta-glycerophosphate (I've also trialled 2mM B-GP and 10mM B-GP and did not notice any immediate differences).
I then culture cells for 3, 6, 9 and 12 days in this osteogenic media washing and fixing them at their respective time points.
Alizarin red protocol:
Once all time points have been fixed, I wash the cells twice with DI water and then add 1ml of 2% alizarin red (pH ~4.2) to the well and I've tried this staining for 5 minutes and up to 30 minutes, I then wash the wells 3 times with DI water.
I tend to see an overall staining of the well with small red dots showing in the wells. I am of course looking for nodules as other papers have indicated however I still see a bit of background and the nodules are not as intense nor as large as you'd see in publications (so it would seem). The contrast between the background and the specific dots is not as clear as it is shown in publications.
Upon inspection under EVOS XL Core microscope there are definitely nodules of cells where the stain is gathering in a more intense manner, suggesting the staining is specific, despite the overall culture well looking red.
Many publications have great images with clear nodules and minimal background whereas others have less defined clear staining and I'm unsure which I should use as a guideline.
While culturing the cells I definitely see cloudy areas which indicates to me a collagen matrix has been synthesised, before staining the cells I also seen black-ish looking deposits which I think may be mineral.
I am also detecting collagen 1 and alkaline phosphatase at the RNA and protein level (Col1 protein increasing over the time course) and osteocalcin RNA expression increasing over time suggesting something is definitely going on.
Any information or advice would be appreciated.
Thanks,
David.
Hi David, I usually wash the cells with DI water as many times is needed to reduce the background after the staining. I have repeated the washing process for up to 6 times. I would also recommend you to do Von Kossa staining, it gives you much less background. You can also decrease the initial cell density or Alizarin Red concentration, I use 40 mM (my protocol is attached). Good luck!
Hi all, thank you for your answers!
I would just like to check; how would cell seeding density impact the mineralization process? I'm needing to wait until the cells are conlfuent before I begin the mineralization process, so how does seeding less cells help with this?
Thank you again for your time.
Hi David,
We would typically also use dexamethasone (0.1uM) in osteogenic induction medium. I would also try and avoid fixing with PFA/formalin as it can have anegative effect on the adjacent wells, 95% methanol for 10 minutes will fix and then overlay with PBS for the remainder of the experiment.
Alizarin red staining is generally pretty straightforward, there is usually some background but nodules are generally much brighter and also quite refractive. 10 minutes in the stain is sufficient and I would also recommend washing with tap water rather than DI water as you get a much brighter red rather than a more orange colour. Do you have any images we could look at?
Tina
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