Need advice for growing THP-1 cells - (Nov/02/2007 )
Dear fellow cell culturists,
After growing many suspension cultures over the years, I am now growing for the first time some THP-1 cells and would like some advise as they seem to grow a little differently.
First ... I am using RPMI 1640 media with 10% FCS, 2 mM L-glu and 0.05 mM 2-ME. I initially seeded the cells at 1 x 10e5 cells/ml and after 5 days, they reached an incredible (being sarcastic) 2.98 x 10e5 cells/ml. I then read the ATCC information sheet and realised they do better at a lower seeding density so tried 5 x 10e4 cells/ml and after 4 days they reached 2 x 10e5 cells/ml, which was better.
So question 1: what is a good range to seed cells for maintaining the culture?
Second ... I need to look at MAPK phosphorylation in these cells and I normally serum-starve the cells overnight before treating. THP-1 cells adhered after serum-starvation 
so question 2: does anyone know the lowest concentration of serum that will keep these guys in suspension?
Finally, I need lots of cells to perform my experiments and since they grow at low densities, I am trying to work out best way to expand them.
So question 3: What type of culture vessels do you grow these cells to get them to high densities?
any advise would be greatly received.
AussieUSA 
FBS? Hm, when I do my experiments I never use FBS in my media. I change the media 24 hours before treating them and after 72 hours they are still in suspension.
I use flasks like this: example flask
Mostly I perpare 25 ml of medium then add 5 ml of cell suspension and let them grow for 2-4 days.
THP-1 cells differentiate into macrophages very easily. When they start to adhere, they've already differentiated. Use heat-inactivated FCS instead, the normal FCS will cause differentiation. I'm using 10% heat-inactivated FCS and my cells are growing well in suspension. Well... I hope it works for you as well. =)
After growing many suspension cultures over the years, I am now growing for the first time some THP-1 cells and would like some advise as they seem to grow a little differently.
First ... I am using RPMI 1640 media with 10% FCS, 2 mM L-glu and 0.05 mM 2-ME. I initially seeded the cells at 1 x 10e5 cells/ml and after 5 days, they reached an incredible (being sarcastic) 2.98 x 10e5 cells/ml. I then read the ATCC information sheet and realised they do better at a lower seeding density so tried 5 x 10e4 cells/ml and after 4 days they reached 2 x 10e5 cells/ml, which was better.
So question 1: what is a good range to seed cells for maintaining the culture?
Second ... I need to look at MAPK phosphorylation in these cells and I normally serum-starve the cells overnight before treating. THP-1 cells adhered after serum-starvation
so question 2: does anyone know the lowest concentration of serum that will keep these guys in suspension?
Finally, I need lots of cells to perform my experiments and since they grow at low densities, I am trying to work out best way to expand them.
So question 3: What type of culture vessels do you grow these cells to get them to high densities?
any advise would be greatly received.
AussieUSA
hi I have been using THP-1s for my cytokine work, and they appear to be healthy so far after around 20 passages. (I might not be that experienced since I am a UG currently)
I grow them in RPMI 1640, 10% HI FBS, 2mM L-Gln, 1x NEAA, 1x Pen/Strep mix. I am using 175ml Falcon flask and medium volume is 75ml total. I am keeping them in 37C, 5% CO2 humidified atmosphere.
I usually seed them at 5 million population. Splitting them every 4 days, they grow to around 50 million before splitting.
hope this helps.
Yes, i also never see them adhere after serum starvation, and they even continue to proliferate somewhat in SF media. As the other guy said, is your FBS heat inactivated?