4 replies to this topic

[BryanC]

Hi.

Im fairly new to cell culture.  I have been working with THP-1 cells.  Its a suspension culture but upon inducing differentiation they attach pretty strongly.  I am running into trouble when I harvest cells for RNA isolation.

I seeded the cells with various induction medias at 300K/ml density and 3e6 cells total in T75 flasks.   I have attempted to detach the cells by incubating in PBS with 5mM EDTA at 37°C as per literature sources, but the cells do not attach even after 30 minutes.  I ended up scraping them with a cell scraper.  My RNA yeild and quality were not very good(RNeasy kit and nanospec), is this a product of scraping the cells?  I should be getting more, as well as better quality RNA.  The same number of cells harvested from un-induced cultures(suspension) provide much more and higher quality RNA.

Any suggestions for a better way to lift the cells, or a different approach to induction that would lead to better/more RNA.

Any help is appreciated.

Edited by BryanC, 19 May 2010 - 11:40 AM.

[jakatta70]

Hi Bryan,

When I used to extract RNA from THP-1 macrophages in 12 well plates I never detached them from the plastic. I simply added 1 ml of TRIzol (Invitrogen) and left at 4 degrees celsius for 10 min. I then extracted the RNA either the old fashioned way using chloroform, ethanol etc or by using the RiboPure kit from Ambion (I recommend this kit because of its ease of use and rapid, problem free process). I obtained yields of up to 200 ng/ul and 2.1 purity (NanoDrop spec) from 250,000 cells/ml. You might think this is low but all I needed was enough to make a solution of RNA at 10 ng/ul for qPCR purposes.

Give this method a try and see if it improves your results. I believ you onyl need to detach the cells carefully if you want to use them for things like Flow Cytometry.

[BryanC]

jakatta70, on May 19 2010, 04:07 PM, said:

Hi Bryan,

When I used to extract RNA from THP-1 macrophages in 12 well plates I never detached them from the plastic. I simply added 1 ml of TRIzol (Invitrogen) and left at 4 degrees celsius for 10 min. I then extracted the RNA either the old fashioned way using chloroform, ethanol etc or by using the RiboPure kit from Ambion (I recommend this kit because of its ease of use and rapid, problem free process). I obtained yields of up to 200 ng/ul and 2.1 purity (NanoDrop spec) from 250,000 cells/ml. You might think this is low but all I needed was enough to make a solution of RNA at 10 ng/ul for qPCR purposes.

Give this method a try and see if it improves your results. I believ you onyl need to detach the cells carefully if you want to use them for things like Flow Cytometry.

Thank you for your reply.

For now, I only need RNA from the cells.  For starters, I'm basically just trying to differentiate to various polarizations (activation states) and trying to develop an assay via pcr to identify the states correctly.    I will eventually need to use the cells for Flow as well, so I will need to be able to efficiently detach them with minimal damage, but for now I just need RNA to see if I can correctly induce and identify the various activation states via pcr.  

So, for the RNeasy kit, could I just add the RLT lysis buffer directly to wells if I induced on plates instead of in flasks?  

As far as your mentioned [] and purity, that would be plenty good enough.  The RNA I isolated from un-treated and cytokine only(no PMA treatment) samples were over 2.0 260/280, and 200-400 ng/ul.  However, my samples from anything I had to scrape to detach was 25-40 ng/ul and 1.5-1.8 260/280.

Edited by BryanC, 19 May 2010 - 02:09 PM.

[jakatta70]

Hi Bryan,

I used to run similar experiments to you with the macrophages. I was looking at classically and alternatively activated states using either cytokines or parasitic helminth extracts.

Yes you should be able to add the RLT buffer directly to the wells of the plate. The cells should burst open and you can then pipette the supernatant into a pre-chilled sterile tube on ice. Do everything in this extraction process on ice to slow down RNA degradation.

I thought I had a protocol saved for detaching THP-1 cells but I don't (only one for detaching Caco-2 intestinal cells). However the protocol might be similar to that used for intestinal cell lines (trypsin edta-pbs). If you check online or on NCBI you should be able to find a protocol easily.

[BryanC]

jakatta70, on May 20 2010, 04:15 AM, said:

Hi Bryan,

I used to run similar experiments to you with the macrophages. I was looking at classically and alternatively activated states using either cytokines or parasitic helminth extracts.

Yes you should be able to add the RLT buffer directly to the wells of the plate. The cells should burst open and you can then pipette the supernatant into a pre-chilled sterile tube on ice. Do everything in this extraction process on ice to slow down RNA degradation.

I thought I had a protocol saved for detaching THP-1 cells but I don't (only one for detaching Caco-2 intestinal cells). However the protocol might be similar to that used for intestinal cell lines (trypsin edta-pbs). If you check online or on NCBI you should be able to find a protocol easily.

Thanks for the suggestions.  I think I will try inducing on plates and lysing directly.  

As far as getting the cells to detach with minimal force, I will definitely need to be able to do this once I am comfortable with the pcr assay and move forward with experiments.  

Some questions for you, or anyone:

1)  Can I increase the [EDTA] I use to lift the cells.  How much is too much?

2)  I can't find the source right now, but I read about someone using 5mM EDTA in PBS but placing the cells on ice during this step instead of 37°c.  Does anyone have any input for this method?

3)  Is there any reason to not use trypsin?

Edited by BryanC, 20 May 2010 - 06:51 AM.