We have some human frozen whole bloods available to us (+DMSO). I would like to thaw them and run a whole blood cytokine stimulation assay +/- LPS +/- dexamethasone. How should I get rid of the DMSO. Is this a factor to worry about?
hello, please see these papers for methods.
http://cebp.aacrjournals.org/content/16/10/2160.long
http://www.ncbi.nlm.nih.gov/pubmed/12433734
See also this short review:
http://www.dmso.org/articles/pharmacology/CellularMolecularAspects.pdf
***
DMSO is good cryoprotective agent that promotes conservation of cellular integrity during freezing and storage.
If your experiment does not involve cell culture after thawing, but involves centrifugation, fractionation or even lysis (and then biochemical analysis including ELISA)-- meaning it involves virtually no time for DMSO to assert an effect on cytokine production and release -- then DMSO should not be a huge concern.
5% DMSO is normally sufficient to keep cells in good state (esp. coupled to the use of Mr Frosty type of tools to gradually freeze samples slowly: http://www.sigmaaldrich.com/catalog/product/sigma/c1562?lang=en®ion=HK)
The references above use 10% DMSO.
***
In viability tests (leukemia cells), we found that DMSO at 5-10% affect growth rate (slows it down), but not to the extent of causing visible cell death. It is known however, that DMSO alters membrane permeability, and thus surface signaling and adhesion could get affected. That's for cell culture, where it becomes a concern.
Good luck.
For the non-cellular fraction, you can assess cytokine contents after clearing (centrifuge) of insoluble fractions (including cells) and sufficient dilution. DMSO would not be an issue for non-cell contents.
***
As for the cell pellet, you can obtain by low -speed (50-100x g) centrifugation of sufficient time (this you need to consult established protocols). Then wash the pellet by resuspension in a suitable medium (some use buffers like HBSS) -- after separation with the supernanant (serum).
Repeat this wash-centrifuge step to dilute out DMSO.
For leukemia cell lines like THP-1, this dilution step is regularly performed upon thawing of the cells from liquid N2. DMSO needs to be removed, to avoid non-specific effects on growth and signaling.
It should be doable.
Thee are a number of WB stimulation assays available, however, none describing the procedure after cyroprotection of samples. What I propose to do is thaw the samples, which have DMSO in them. Then spin them down, aspirate plasma. Resuspend in RPMI 1640 or equivalent. Then spin down again, and aspirate medium. Then I would start the WB stimulation assay. What do you think about this approach?
Hello, that's broadly similar to what I mentioned. I anticipate that the primary blood cells may be a bit fragile after thawing, so I suggest that moderate centrifugation conditions (low speed, sufficiently long time) be used.
You can have a few controls, after this removal of DMSO (essentially by serial dilution), in which a small amount of DMSO is re-added, then assess the cytokines of interest to you, and compare the results with groups with no DMSO re-added.
0.3% DMSO is the max level of the solvent considered "okay" in in vitro drug screens. Careful design would try to reduce it further to like 0.1%, which is possible by adding a drug stock solution (1 uL) to a 1 mL volume by 1000x dilution.
The serial dilution step certainly should reduce the residual DMSO amounts to
- Badges
- Science topic