I am trying to freeze the bone marrow extract from rats. In a reference protocol, the centrifugal speed is about 450 g in 5 min. But my lab only has a centrifuge with a fixed speed of 980 g.
I did centrifuge the bone marrow at 980 g in 5 min in culture media solution. Does it really affect the cells' adhesion and viability? Thanks for your answers.
Hi Long, in my experience, hard centrifugation of cells makes a more compact cell pellet without affecting cell viability or function.
But, if you want to be sure that this centrifugation does not harm your cells, do a trypan blue stain of your cells before and after the spin.
Can’t add (now) perhaps not much still, but IMHO (at least for making buffy coats from peripheral blood –containing lymphocytes within an upper layer of platelets and a lower layer of RBC’s for further TEM-examination) the difference between 450 and 980 g (5 min) for peripheral blood components will not make a serious difference (at least for preservation of ultrastructure when the buffy coat is fixed and processed state of the art). There the optimum is said (and my own experience for years) to be 1000 g for 5-10 min (cooling to RT or 36 °C during separation, 450-500g/10 min, 1000 g 5 min sufficient).
Questions arising: which kind of rotor you have on your centrifuge? Cooled-non-cooled? What about ROS-activity? Do you expect perhaps harmful increase in ROS activity with your BM-cell fraction? (for semen [article Shekarriz et al. from 1995] it has been reported that there might be an comparing between 200g/500g – for 2 or 10 min centrifugation: esp. if centrifuged for 10 min, regardless of g-force, so ).
Thinking of using density gradient centrifugation on Ficoll-Paque (for BM-cells): not knowing whether this could be of help to you, cf.
Radrizzani et al. Journal of Translational Medicine 2014, 12 :276
http://www.translational-medicine.com/content/12/1/276
at least for the yield authors found there is not much difference between 400 g 20°C, 30’ and 719 g 20°C, 20’. In the end I think, keeping temperature during centrifugation, choosing the right rotor, the right medium and perhaps centrifugation as short as possible (if you don't have a cooled centrifuge) will be of benefit for your BM-cell preparation.
Good luck, and best wishes.
Thanks so much for your answer Wolfgang. It actually adds another point I need to think about in my experiment.
I don't know if ROC-activity can really affect MSCs at 980g for 10 min. But maybe, I should take this into account to optimize the centrifuging time to attain the best cell viability.
I have a question: how the rotor is affecting the cells more than the force? I am using IEC 221 rotor of 13 cm in arm length. I thought that in centrifuge, the force is more important, isn't it?
My experience is that centrifugation speeds under 4000 rpm for 5-10 minutes do not change the percentage of bacterial cell viability. This is my experience, I can recommend it to try.
Dear Long Pham, you're welcome.
So I guess you are usíng a Thermo(?) 221 6 x 15mL Economy Swinging Bucket Rotor
(Thermo Scientific™ 221 6 x 15mL Economy Swinging—Bucket Rotor is a low cost rotor for 10-15mL tube) - See more at: http://www.thermoscientific.com/content/tfs/en/product/221-6-x-15ml-economy-swinging-bucket-rotor.html#sthash.yrabfFJz.dpuf
This is a "swing-out"-bucket rotor which IMHO is for sure suited for your task. For further "basic reading" on centrifugation (if you nedd that) I would like to point you to: at http://www.coleparmer.com/TechLibraryArticle/30. There you can find also:
Affection of rotor type versus g-force: IMHO (besides see above about ) it is important that your rotor runs vibration-free (i.e. no defect bearing of the rotor) and if the centrifuge has a "break": don't use it... From a swing-bucket rotor spun vial IMHO you can harvest your cells easier than from a vertical or fixed angle rotor spun vial, especially when you don't "fix" your cells with a fixative.
Hope this helps, best wishes and regards, Wolfgang
if cells broke down during centrifugation how would we figure that out?
Hi Naila,
Any cell viability assay will work. The most common (and cheapest) is Trypan Blue which is excluded by live cells so only the dead cells turn blue. Methylene blue is another common cheap dye. Just mix the dye 1:1 with you cell aliquot, count on a hemocytometer and multiply by 2 to account for the 1:1 dilution. If the difference in viability is very dramatic you may not even need to count them.
Best of luck!
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