6 replies to this topic

[Elioelio]

I am harvesting adherent cells from a 24 well plate - there should be at least 100,000 cells in each well. I use trypsin and then add 1ml media and put into eppendorf tubes.
I then centrifuge for 5000rpm for 5 mins. (this is is a 6cm radius centrifuge). However, I do not see a pellet of cells. I remove the supernatant anyway, as I know where the pellet should be, and then resuspend in 200ul media. This is then added to a 96 well plate for flow cytometry. I can see under the microscope there are cells there. However, flow cytometry shows me that there are usually only 2000-3000 cells.
I'm guessing the other cells didn't pellet (I checked, they are not still adhered to the well).

When I pellet cells in 15ml tubes from T75 flasks for passaging and counting, they pellet fine, and this is at a much lower xg. (1000rpm, 5mins, in a larger centrifuge).
5000rpm is very fast - I know it it probably too fast. So why is it that not all the cells are pelleted?

Any idea on how to redress this?

Thanks for any help or ideas!

[leelee]

Perhaps the faster centrifuge speed is rupturing and destroying the other cells? I would use the same rcf as you would typically use for your larger centrifuge.

Also, have you checked in your wells after trypsinisation to make sure you are in fact recovering all of your cells?

[Tabaluga]

I would definitely try with a lower rpm. When you resuspend in media, do you "wash" it down the wall of the Eppi tube ? Because there may be cells smeared against the wall. Also, are you sure the majority of cells don't get lost during your FACS staining procedure somehow ?

[ff11]

I agree, if you refer to the table in the following link: http://www.piercenet...ifuge-speed.pdf
by going from 1000rpm to 5000rpm, at a radius of 6cm, you are increasing the centrifugal force from 67g to 1,677g.

[shirosands]

first, I agree you need to check the well after you remove the cells to verify you really removed them.

2nd, how much trypsin are you using and how long do you leave it on?

pretty much, observe the cells in the well when the trypsin is on and verify they are detaching and you are then removing them.

[ioannap]

Hi there,

I agree with all that is mentioned above regarding the speed, 5000rpm is probably too much, however one more thing you could check is that all this does not have anything to do with your 1.5ml centrifuge tubes.

Is anyone else in your lab having similar issues?
At some point we had this problem in our lab and were very confused with not getting the expected pellets.
We had recently changed the supplier we had for the tubes. We connected the dots finally and tried harvesting cells from 2 duplicate wells and pelleting them using tubes from our previous and new supplier.
There was a nicely formed pellet in the "good" tubes and almost nothing visible in the "bad" tubes. There were cells smeared on the wall of the tube, but that's not good enough really.

If in your case it has anything to do with the tubes, I would suggest -if you cannot change the tubes obviously!- to spin at lower rpm and for longer, like 6-8mins, which seemed to help a bit.

Good luck!

[angelafuertes]

Are you filtering your samples for flow-cytometry? If so, check that the cell strainer pore size is not too narrow (or your cells are not clumped), otherwise your cells will get stuck there and you will end up with near nothing (actually happened to me). Perform a little test: count your cells with a hemocytometer before and after filtering.