4 replies to this topic

[OKSO]

Hi,

I have been working with 'normal' THP-1 monocytes in culture for some time, and always spin them down at 1,000rpm for 5 min.

Now, I am starting experiments with THP-1-Blue-CD14 cells, which are stably transfected with a reporter plasmid expressing an alkaline phosphatase gene. The protocol from the supply company states that these cells should be centifuged at 3000 - 5000 rpm for 5 mins.

This seems excessively high for any cell line .. does anyone have any experience of using this transfected cell line, and if so do you spin them down at this speed?

Thanks!

[gfischer]

OKSO, on Jul 23 2009, 05:11 AM, said:

Hi,

I have been working with 'normal' THP-1 monocytes in culture for some time, and always spin them down at 1,000rpm for 5 min.

Now, I am starting experiments with THP-1-Blue-CD14 cells, which are stably transfected with a reporter plasmid expressing an alkaline phosphatase gene. The protocol from the supply company states that these cells should be centifuged at 3000 - 5000 rpm for 5 mins.

This seems excessively high for any cell line .. does anyone have any experience of using this transfected cell line, and if so do you spin them down at this speed?

Thanks!

I would recommend calling the company and finding out the diameter of the rotor they used or the relative force.  The rpm itself is inconsequential, it's the force involved that can damage the cells, and the force is dependent on the size of the rotor in addition to the rotation speed.

[OKSO]

Thanks gfischer, thats a good point.
Cheers!

[SuMi]

Hi, I have used THP-1 blue cells and the protocol for resuspension also says to centrifuge them at 3000-5000rpm. We spun our cells at 1500rpm and they were fine.

[OKSO]

Thanks SuMi,

Yes, I think I will spin them at my normal 1,000 rpm.
THe company said to stick to my normal protocol - which makes more sense to me anyway
Cheers!