Hi Yanny,

Could you please be a bit more specific about the condition of your experiment.

Judging by your topics of interest, I assume you are growing cells on HA.

Regardless of the shape of HA being used, I would suggest that prior to lysing the cells, you should harvest the cells from the surface of the HA particles, using several consecutive trypsin treatments.

The effectiveness of this approach can be determined by monitoring the number of cells collected in each fraction, which can be done using a simple cell count (Trypan Blue exclusion assay).

You could also check the extent of cells that remain attached to the HA after a series of treatments, by processing the HA particles and viewing them using SEM.

You could also do DNA quantitation on both HA particles and the harvested cells suspension after each trypsin treatment and establish whether you have managed to collect as much of the cells as possible (you may not be able to collect all of the cells).

I would also suggest a brief centrifugation step of the pooled fractions of cells suspension, to remove any HA debris, transfer the cells suspension to a new tube followed by DNA extraction.

I hope this is helpful.

Basil

Hi Basil,

Thank you for your reply. Yeah, I was thinking to trysinize the samples too. I'm doing cytotoxicity test of hydroxyapatite powders on MG63-osteoblast-like cells. I found that the DNA quantities varies so much between each timepoint. I suspect that something wrong with my DNA assay, most likely the HA particles bound to the cells and thus giving me the false reading of DNA quantities. I found a paper saying that HA particles are charging particles and tends to bind the cellular DNA. It is suggested that the lysate should be trypsinized before freeze them down followed by thaw.

Are you working on materials and cells? I have another question actually. If let say I trypsinized the samples, would it affect the total protein assay as I'm using the same samples? Will the use of trypsin affect the amount of total protein? I'm doing Bradford Total Protein to be exact.

Thanks,

Yanny Marliana

Hi Yanny,

Yes, my research is on biomaterials. I don't see why using trypsin would affect your protein assay. You will be harvesting the cells, neutralize the trypsin by adding serum-containing medium, then you will centrifuge the cells suspension, lyse the cells pellet and then measure their total protein.

In your previous question you mentioned that you are trying to determine the materials suitability to differentiate hMSC to osteogenic lineage, not MG-63.

Whichever cell type you use, I would recommend doing an indirect study initially, using multi-well plate with inserts. This would remove or minimize the problem of dealing with HA powders, but that is something that you need to discuss with your research manager.

Best of luck Yanny, I hope all goes well

Regards

Basil

Hi Basil

Thank you very much. I'm currently working on both cell line, hMSCs and also MG63. Yeah I have done some reading, looks like trypsin would not affected my samples for protein. Thank you again. Best of luck to you too :-)

Would you please share the reference of the paper that states that HA binds DNA?

Dear Juliane,

Here are some papers highlighting Hydroxyapatite's ability to bind DNA, and its use in gene delivery:

1) http://cdn.intechopen.com/pdfs/23626/InTech-Transfection_of_bone_cells_in_vivo_using_ha_ceramic_particles_histological_study.pdf

2) http://www.ncbi.nlm.nih.gov/pubmed/22834406

3) https://melbourne-systems.s3.amazonaws.com/asset/file/51162f9257817e5a160000a2/Brundin_2013.pdf

Regards

Basil