Culturing THP-1 cells, they grew too slow...Need help... - (May/27/2008 )

Hi, I am culturing THP-1 cells (ATCC). Currently they are P14 and P15. I was told to thaw them at P13,P15 for trail and this is my very first time culturing THP-1.

The media consist of
- RPMI powder (10.3g /L)
- 2g/L sodium bicarbonate
- 2mM L-glutamine
- 10% FCS (Heat-inactivated)

All the following components are filtered through 0.2um membrane. They are growing but they seem to grow very slow. According to ATCC, the doubling time is 26hrs... Anyone know of any way to speed up the growing?

Many helps appreciated...

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if you use media according to what was recommended, should be OK. Try do a growth curve to determine doubling time. if the doubling time very much difference from 26 hrs, maybe you should consider not to use the cells. Your cells might have some problem.

I don't recommend to add anything to SPEED UP the growth. It might alter the cells characteristic.

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Are they growing yet?

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Hi there

We grow THP-1 cells and add 2-mercaptoethanol to a final concentration of 0.05 mM (as suggested by ATCC). We also keep a close eye on them: Subcultutre when cell concentration reaches 8X105) cells/ml.Do not allow the cell concentration to exceed 1 X 10(6) cells/ml.

We determined their doubling time - but I have that data at work. PM to remind me if you want to know

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There! I knew Clare would have the answer

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[/quote]
There! I knew Clare would have the answer
[/quote]

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Yep. They are still growing but at a slow rate. Since THP-1 are floating cells, how I can differentiate between the dead and the alive cells? I tried to centrifuge them at 1,400 rpm for 10mins at room temp to remove the contamination. Does centrifugation at this speed really helps to remove contamination?

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The old fashioned way of getting rid of dead cells is to:

Allow the cell suspension to settle for upto 2 minutes. The live cells are more dense and will sediment quicker than the ghost/dead cells. You will lose some viable cells however, but the majority of dead cells will still be floating in the media ad can be discarded .......CAREFULLY.

Kindest regards

Rhombus

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Sorry forgot something:

1,400RPM for 10 minutes is far too fast to spin cells.

100g for 5 minutes is ample for sedimentation

Rhombus.

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What for you add b-mercapto to THP-1 cells? What happend to THP-1 cells If I don't add it.

I have problems with THP-1 cells too. Some of the cells attach to the surface: are they activated cells? How can I avoid that?

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