THP-1 culture protocol - (Dec/01/2009 )

Hi everyone,

I worked with the human monocyte line, THP-1, in my first postdoc and never had any problems with it. The only "problem" I had was having too many cells! Im now in a postdoc position elsewhere and I purchased THP-1s from ATCC. The first vial simply didn't grow and then the numbers of cells started to decrease significantly after every centrifugation (120 g for 5 min) presumably because the dead cells floated away into the media. Although I was confident that the cells weren't dying because of my aseptic tenchnique, I purchased a new vial anyway. Again the cells have died!! Im very frustrated.

Here is the composition of my media: RPM1 1640 containing 2 mM glutamine; 10 mM HEPES; 1mM sodium pyruvate; 4.5g/L glucose (25 mM; can someone check that the molarity I have stated is correct for 4.5g/L glucose?); 1.5g/L sodium bicarbonate (18 mM; again can someone check?); 10% FCS; 0.05 mM beta mercaptoethanol.

I thawed the vial, cleaned it, transferred the cells to a 50 ml tube, added 9 ml of media dropwise, centrifuged at 120 g for 5 min; removed supernatant; resuspended in 1ml of media and seeded at 200,000 cells/ml in a T25 flask for 7 days, adding media every 3 days. Now I must say that this isnt the technique I have used previously. I used to add 30 ml of media to the cells and let them sit overnight at 37C before centrifuging them. In addition the media composition advised by ATCC is different than what I used before. I added 17.5 ul of 1M HEPES, 10 ml of penicillin streptomycin, 10% FCS.

Can anyone advise me on what Im doing wrong? Has anyone had similar problems with THP-1s from ATCC?

Many thanks.

Michael.

I'm currently working with THP-1 cells and having the same problems. I recently ordered a batch from ATCC (which took three weeks to arrive here in Sydney). Followed the instructions ATCC provides exactly (same techniques you used) and the cells weren't growing. After my inital thawing of the cells, I did a cell count and assessed the viability using trypan blue exclusion. There were very few cells, and even fewer cells alive.

I contacted ATCC, and the only suggestion they had was to use different FBS (I was using qualified, not certified, serum, which I have been using previously on THP-1 cells with no problems) and not heat inactive my serum (which I have again been doing with no problems).

Also, it seems that not all THP-1 protocols use the 0.05 mM 2-ME, but ATCC says it's necessary. THP-1 cells from Sigma-Aldrich come in 10% glyerol as the cryoprotectant, but ATCC uses 5% DMSO. It is pretty well established in the literature that DMSO (although at higher concentrations, around 20% I believe) causes THP-1 cells to differentiate into macrophages. I asked ATCC about this, and they said they couldn't comment on it. It seems like everyone gets their cells from ATCC, so I'm assuming the 5% DMSO isn't a problem (or at least it is a consistent problem for everyone working with ATCC THP-1 cells).

I'm in the process of purchasing another vial of cells from ATCC, and hopefully these will grow. I have also talked to a few other researchers who regularly use ATCC THP-1 cells, and they have reported "bad vials" that spontaneously differentiated, didn't grow normally, or just died, so it seems that this is a common problem.

Good luck with ATCC and getting a better vial.

Hello,

I'm new in the discussion, but I also have worked with THP-1 cells during my postdocs without any problem in growing them. Now I started in my lab, with the same protocol and media and the cells don't grow. The supplier mantained the cells frozen with 10% of DMSO and told me I should grow them with the 20% of FBS instead of 10% I used regularly. As you comment the THP-1 batch used before come from cells frozen with glycerol media. Seems that we had similar problems. I will explain if I had progress with them.

Arale

Hello,

I'm starting with THP-1 culture, I need macrophages, so I use PMA to differentation of them. I would like to know If I can store my RPMI medium with PMA in the refrigerator for a few days?

Anula

Anula on Mon Oct 3 08:39:08 2011 said:

I store working concentration RPMI+PMA in the -20C for weeks at a time and the cells still differentiate over 24-72 hours. 4C might not be such a great idea, but I don't have empirical experience to back this claim up.