12 replies to this topic

[Tabaluga]

Hi,
when I centrifuge cells (1300 rpm, 3 min) in a small 1.5 ml reaction tube, a lot of them always stick to the wall of the tube (the outer part, due to the centrifugal force). This is irritating because it means a lot less cells in the pellet !! Higher centrifugation speed or time does not solve the problem.

Do you have suggestion or advice how to get rid of this problem ?

Thanks

[casandra]

Tabaluga, on 07 January 2013 - 10:47 AM, said:

Hi,
when I centrifuge cells (1300 rpm, 3 min) in a small 1.5 ml reaction tube, a lot of them always stick to the wall of the tube (the outer part, due to the centrifugal force). This is irritating because it means a lot less cells in the pellet !! Higher centrifugation speed or time does not solve the problem.

Do you have suggestion or advice how to get rid of this problem ?

Thanks

what kind of microfuge tubes do you have? Use of polypropylene tubes would minimise this sticking.....if it's really a problem, some people coat the tubes with BSA (~5%?...but of course, if it doesn't interfere with whatever you will do with the cells later on)....

[Tabaluga]

We normally use 1.5 ml Eppendorf safe-lock tubes, I don't know what material they are made of. Coating would really be lots and lots... and lots of work given that I use them extremely often........

[casandra]

Tabaluga, on 07 January 2013 - 11:54 AM, said:

We normally use 1.5 ml Eppendorf safe-lock tubes, I don't know what material they are made of. Coating would really be lots and lots... and lots of work given that I use them extremely often........

ok, I think those are polypropylene...you centrifuge them in which medium/buffer? Couldn't you just "wash" (gently) the walls up and down to maximise your recovery?

[Tabaluga]

Usually I centrifuge them in PBS/1%BSA. You mean wash them down and centrifuge again? Certainly, that would be a good thing to try. Problem is again that it is very time-consuming when i have lots of tubes and lots of centrifugation steps (for example 7x centrifuging and 16 tubes....) So I was rather searching for something so it does not happen in first place

[pito]

Tabaluga, on 07 January 2013 - 01:00 PM, said:

Usually I centrifuge them in PBS/1%BSA. You mean wash them down and centrifuge again? Certainly, that would be a good thing to try. Problem is again that it is very time-consuming when i have lots of tubes and lots of centrifugation steps (for example 7x centrifuging and 16 tubes....) So I was rather searching for something so it does not happen in first place

yeah, buy other tubes...

THere is nothing else you can do: coating the tubes yoursels is very time consuming so just buy other ones (more expensive ones).
You have do to it sometimes.
I have to do it too, so I just buy non sticky tubes.

[casandra]

Tabaluga, on 07 January 2013 - 01:00 PM, said:

Usually I centrifuge them in PBS/1%BSA. You mean wash them down and centrifuge again? Certainly, that would be a good thing to try. Problem is again that it is very time-consuming when i have lots of tubes and lots of centrifugation steps (for example 7x centrifuging and 16 tubes....) So I was rather searching for something so it does not happen in first place

7x centrifugation with 16 tubes (new each time?)? Ok that's a lot of tubes and what I actually meant was not to recentrifuge but just to manually rinse the walls by gently pipetting up and down with resuspension buffer to loosen up the stuck cells...however, with the number of tubes you're handling, this would be tedious, time consuming and a surefire way to get carpal tunnel syndrome faster ...so yup, better to ask for samples of low-binding, non-stick microfuge tubes from Costar or Eppendorf etc for testing....

[bob1]

You can also spin at lower speed for longer to get the cells to form a single pellet rather than smear up the wall of the tube.

[Tabaluga]

Thanks everyone for sharing your ideas !

@pito: That's true, but I see this as my last resort. I don't think the lab will buy more expensive tubes because it's weird, I seem to be the only person having this problem (my cells perhaps ??)

@casandra: I already try to pipet the rest of the stuck cells down when resuspending, but somehow the cell number still diminishes greatly. Maybe due to shear forces when I remove the supernatant with the pump or something like that ?

@bob1: Good idea, I never tried that. I can try this out tomorrow and will certainly report back whether it worked.

Edited by Tabaluga, 08 January 2013 - 12:48 PM.

[casandra]

Yup, bob's suggestion might work. Do you have a tight pellet or a fluffy one after 1300 rpm centrifugation? bec if you think that you're vacuuming off some of your cells then either leave a little bit of supernatant or or just pipet off manually  (which will once again lead to an early carpal tunnel syndrome )...you need new bionic hands ....

[pito]

Tabaluga, on 08 January 2013 - 12:44 PM, said:

Thanks everyone for sharing your ideas !

@pito: That's true, but I see this as my last resort. I don't think the lab will buy more expensive tubes because it's weird, I seem to be the only person having this problem (my cells perhaps ??)

@casandra: I already try to pipet the rest of the stuck cells down when resuspending, but somehow the cell number still diminishes greatly. Maybe due to shear forces when I remove the supernatant with the pump or something like that ?

@bob1: Good idea, I never tried that. I can try this out tomorrow and will certainly report back whether it worked.

But adding substances to prevent sticking, costs money and time too..

You could do what bob1 said, but I wonder if you could collect everything of your sample in an easy way like that.

Its always a question about whats best in the end: buying more expensive tubes or messing a long time trying to avoid buying them.

[Tabaluga]

@ casandra: Well I wouldn't say it's fluffy but it can move around if you aspire the supernatant close to it. Also i always leave a tiny bit of supernatant as to avoid that, however I cannot avoid the cells on the wall getting under "vacuuming stress", at least I think that's where the problem may be. And I already have to pipet off manually a lot in the later parts of my procedures when I use formaldehyde or other fixatives under the fume hood... luckily no carpal tunnel syndrome yet though

@ pito: I know. when other ways fail, I'll see to it that we can test these non-sticking tubes at least. Which ones do you use yourself ?

[Tabaluga]

Unfortunately, bob1's suggestion didn't work for me. So I guess that means I'll have to really look for different tubes... Thanks everyone! If you happen to have any other ideas I'd be grateful to hear them of course !