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cell homogenization


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13 replies to this topic

#1 zienpiggie

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Posted 10 June 2011 - 04:49 PM

Hi everyone,

I have to analyse an enzyme activity from my cell lines, and it appears that I need to homogenize the cells. I don't have those dounce homogenizers or common homogenizing tools. I have read some people sonicate them instead. Would that work, or is there anyone here that has tried other method to replace the homogenization method?

Thank you!

#2 bob1

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Posted 10 June 2011 - 08:36 PM

Could you just lyse the cells in a detergent based solution? Or perhaps use a hypotonic buffer to swell and "pop" the cells?

#3 zienpiggie

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Posted 11 June 2011 - 07:56 AM

Hi Bob1, the enzymes would later then be tested in activity using a commercial kit. I am not sure that chemical lysis may be compatible with the rest of the reagents of the kit. I am thus thinking more of physical method of lysis, such as sonication or freeze thawing, but not sure if anyone can tell me if it will be able to replace the homogenization with those dounce homogenizer or other known homogenizer?

#4 bob1

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Posted 11 June 2011 - 04:14 PM

Well, homogenization with a dounce or similar will disrupt the cells, but freeze/thawing several times should also do it equally well, though this might lead to degradation of the protein.

Sonication will work too, but I am not sure how this affects proteins in the end. I know it can be used for extracting viruses from cells, so it should be OK.

How about using one of those little pestles you get for grinding tissues for RNA extraction in a 1.5 ml tube?

#5 zienpiggie

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Posted 11 June 2011 - 08:54 PM

Hi Bob1, thank you so much for the suggestion. I actually had no idea of how that looked like so I googled it. It looked like this?

http://www.bio-world...able-mL-PK.html

Do you happen to know of how to use them? Like how many strokes to fully homogenize them? I have thought about using QIAshredder for homogenizing the cells, it's those patented microfuge tubes where you just add your samples on top and centrifuge them and you get homogenized samples on the bottom tube, but I think it is going to be really costly since I could only run one sample per tube ($84 for 50 tubes).

I also thought that sonication or freeze thawing would be a lot more time-efficient and economical, but I can see your point about freeze thawing reducing the enzyme activity. I am also worried that using the pestle for manual homogenization may not give as much as uniform treatment as homogenization or freeze thawing?

I think what I need to do is run a small preliminary trial and see the recovery of the enzyme activity with these methods. What do you think?

#6 bob1

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Posted 12 June 2011 - 03:52 PM

Yes, those are the one I was talking about. To use them you keep your sample in as small a volume as possible and just push down and twist to grind the sample. They don't really work quite the same as a dounce, they certainly won't be as precise in the homogenisation. Dounce homogenizers used to be quite common - there are probably still a few around somewhere nearby if you are in a university. Plant people tend to keep these sorts of things...

One thought I had - could you snap freeze on liquid nitrogen or dry ice and then grind the samples to produce a powder? This would only be one freeze/thaw and might be better than sonication or the pestles.

I've never used QIAshredder so can't comment on the effectivness. I suspect that it will just be series of grids overlapping at angles which disrupt the cells as they get forced over them while centrifuging.

I would run a trial like you suggested, there may be some simple and cost effective solution out there.

#7 yimao

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Posted 12 June 2011 - 08:26 PM

hey, i was also looking for a grinder to grind my cell line pellet, and i found this online, is it fit your purpose?

do u know what is the difference between "Large clearance" and "Small clearence" means?

http://www.sigmaaldr...blePage=9578358


Hi Bob1, thank you so much for the suggestion. I actually had no idea of how that looked like so I googled it. It looked like this?

http://www.bio-world...able-mL-PK.html

Do you happen to know of how to use them? Like how many strokes to fully homogenize them? I have thought about using QIAshredder for homogenizing the cells, it's those patented microfuge tubes where you just add your samples on top and centrifuge them and you get homogenized samples on the bottom tube, but I think it is going to be really costly since I could only run one sample per tube ($84 for 50 tubes).

I also thought that sonication or freeze thawing would be a lot more time-efficient and economical, but I can see your point about freeze thawing reducing the enzyme activity. I am also worried that using the pestle for manual homogenization may not give as much as uniform treatment as homogenization or freeze thawing?

I think what I need to do is run a small preliminary trial and see the recovery of the enzyme activity with these methods. What do you think?


Edited by yimao, 12 June 2011 - 08:44 PM.


#8 zienpiggie

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Posted 12 June 2011 - 09:10 PM

Hi Bob1, thank you again for your response. That is a good idea regarding the snap freezing in liquid nitrogen. I remembered a lab mate who tried that though and she used regular microfuge tube and dip it in to my nitrogen dewar. The microfuge popped open right away and she sometimes lost her microfuge tubes in the nitrogen dewar. May be I could scrape my cells, then put them on cryogenic vials instead and dunk it into the nitrogen dewar in a box, and when I'm ready to use them I could take them out. I have never ground my cell samples before though, particularly if the cell sample is going to be in a small amount of cell pellet, which may or may not be in some 'recommended' buffer.. I guess I could have used that pestle you suggested?

@ Yimao, I am not sure what those large and small clearance means. I have never worked with these pestles before so this is new to me too.

#9 zienpiggie

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Posted 12 June 2011 - 09:16 PM

oh wait, you said snap-freeze right. so I have to basically be pretty quick. I dunk it and take it out in split seconds kind of thing. I think I should just go ahead and give it a shot.

#10 rhombus

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Posted 13 June 2011 - 02:33 AM

oh wait, you said snap-freeze right. so I have to basically be pretty quick. I dunk it and take it out in split seconds kind of thing. I think I should just go ahead and give it a shot.


Dear zienpiggie,

The best way of releasing your protein is to SONICATE the cells. I used to prepare enzymes form both mammalian and insect cell lines. The assay required "active" protein and this was achieved using the following protocol:

Resuspend the cells in the smallest volume possible....generally in a buffer that contains multiple protease inhibitors.

Sonicate the cells 3 times for 5 seconds a times using an optimised sonicating power/frequency....this is done on ice and there is a gap of 30 seconds between each sonication.

ultracentrifuge the disrupted cell suspension for 30 minutes at 105,000g

The centrifuged supernatant should contain your active enzyme if cytosolic.



We tried many ways of "Fractionating" the cells i.e. Homogenisation/Sonication/Freeze-thaw ..........and Sonication was by far the best and most reproducible method.

The best sonicator we found was the tried and tested MSE Soniprep Model 150.

We have used this method many times and have published data to support the above:

Biochem and Biophys Res Commun. Volume 188 No. 1, Pages 209-215, 1992 Moncada et al Glucocorticoids do not affect the induction of a novel calcium-dependent Nitric Oxide Synthase in Rabbit Chondrocytes.

I do hope you find this useful.

Kindest regards.

Uncle Rhombus.

#11 zienpiggie

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Posted 13 June 2011 - 03:22 PM

Thank you Uncle Rhombus, I'll definitely try that.

#12 bob1

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Posted 13 June 2011 - 04:59 PM

Rhombus comes through once again...

@yimaio - the clearance large and small is the maximum gap between the sides of the tube and the pestle used to homogenize the samples. With particular size gaps you can rupture the cytoplasm but not the nucleii for instance.

#13 rainlqy

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Posted 05 July 2011 - 11:54 PM


oh wait, you said snap-freeze right. so I have to basically be pretty quick. I dunk it and take it out in split seconds kind of thing. I think I should just go ahead and give it a shot.


Dear zienpiggie,

....

Sonicate the cells 3 times for 5 seconds a times using an optimised sonicating power/frequency....this is done on ice and there is a gap of 30 seconds between each sonication.

ultracentrifuge the disrupted cell suspension for 30 minutes at 105,000g

...
We have used this method many times and have published data to support the above:

Biochem and Biophys Res Commun. Volume 188 No. 1, Pages 209-215, 1992 Moncada et al Glucocorticoids do not affect the induction of a novel calcium-dependent Nitric Oxide Synthase in Rabbit Chondrocytes.

I do hope you find this useful.

Kindest regards.

Uncle Rhombus.



Hi Uncle Rhombus,

I hope I am not misunderstood your suggestion. BUT i still have to ask again, did you mean that the sonication just need 3 times with 5 seconds sonications and 30 seconds between each sonication? That means you just need 5*3+30*2=1minute and 15 seconds to finish sonication.

If I have 1L Hi5 cells, I usually use 100ml lysis buffer, dose such sonication time enough? PS: what is your proper power on your sonicater? Thanks

#14 rhombus

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Posted 08 July 2011 - 07:55 AM



oh wait, you said snap-freeze right. so I have to basically be pretty quick. I dunk it and take it out in split seconds kind of thing. I think I should just go ahead and give it a shot.


Dear zienpiggie,

....

Sonicate the cells 3 times for 5 seconds a times using an optimised sonicating power/frequency....this is done on ice and there is a gap of 30 seconds between each sonication.

ultracentrifuge the disrupted cell suspension for 30 minutes at 105,000g

...
We have used this method many times and have published data to support the above:

Biochem and Biophys Res Commun. Volume 188 No. 1, Pages 209-215, 1992 Moncada et al Glucocorticoids do not affect the induction of a novel calcium-dependent Nitric Oxide Synthase in Rabbit Chondrocytes.

I do hope you find this useful.

Kindest regards.

Uncle Rhombus.



Hi Uncle Rhombus,

I hope I am not misunderstood your suggestion. BUT i still have to ask again, did you mean that the sonication just need 3 times with 5 seconds sonications and 30 seconds between each sonication? That means you just need 5*3+30*2=1minute and 15 seconds to finish sonication.

If I have 1L Hi5 cells, I usually use 100ml lysis buffer, dose such sonication time enough? PS: what is your proper power on your sonicater? Thanks



No problem at all.....you have understood me perfectly:-

sonicate for 5 seconds (on ice) at an optimised power....then let the cell suspension rest on ice for 30 seconds.

Repeat this 2 more times.

Excessive sonication causes heat, and heat will denature your enzyme, thus inactivating it. You have to optimise cell fractionation i.e. release the protein from the cells...check the cell suspension under a light microscope to see that the cells have been fully disrupted.

On the MSE Soniprobe I use, the power rating is in Amptitude microns....and I use 50% of maximum sonication power.....again this level has to be optimised.

Different cells require different disruption powers......so lots of optimisation!!!!

Have fun and good luck.

Kindest regards.

Uncle Rhombus.




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