Dear all,
I already try several months to polarize in vitro differentiated THP-1 cells to M1 and M2 macrophages. I am successful in M0 differentiation and also in polarization. But sometimes cells deattach during the polarization process (48h). Here is the protocol, which I am using:
1. Seeding THP-1 cells (1,3x106 / well) in 6-well plates (2ml RPMI-medium with 3% FCS) and adding 10ng/ml PMA
3. Incubation for 72h.
4. Rest: Washing cells with 1xPBS and adding 2ml RPMI (+3% FCS) without PMA
5. Incubation for 24 hours
6. Polarization in 2ml RPMI medium (+3% FCS):
M1: 1µg/ml E.coli LPS + 10ng/ml IFN-g
M2: 20ng/ml IL-4 and IL-13
7. Incubation for 48 hours
8. Analysis: CD14, CD36, CD11b, CD80, CD206 and CD209
During the second day of polarization cells deattach again (in all wells M0, M1 and M2). And I do not know why. I have already tested lots of conditions:
a. different cell numbers
b. different cell passages
c. new ordered versus "old" PMA
d. I also tested different PMA concentration, different incubation times and resting times for in vitro differentaition (analysing cells after 24h rest, without polarization phase). And I do not want to increase PMA due to its potential to shift THP-1 to M1 macrophages.
I started these experiments with medium containing 10% FCS. Reducing FCS to 3% during the whole experiments is the only conditions which showed significant better results. Using only 3% FCS caused very strong attached macrophages with their typical morphology (not seen when using 10% FCS). However, it worked three times and now it does not work (even with freshly thawed cells).
Could it be a medium issue? For thawing we are using Gibco RPMI with 20% FCS until cells are in exponential growth phase. Afterwards I change the medium to Sigma with 10% FCS (both without antibiotics). When the 3% FCS during experiments caused the significant better results, cells were changed at the day of seeding from Gibco to SIgma medium.
I do not know what else I can check. Has anyone some suggestions??
Thank you very much.
Christian
Hello,
I think the LPS concentration may be the issue, I never used such high concentration, and in the experiments even 100 ng/ml LPS makes cells less viable.
Sorry, didn't notice that all M0-M1-M2 cells are detaching the same day. The second issue may be that the cell density is too high. If yes then probably cells undergo TRAIL-induced apoptosis.
UPD: some simple calculations: 1.3 millions per well of 6-well plate will give around 130 thousand cells per cm^2, which correspond to approx 8 square microns of plate contact area per cell (less than 3x3 um spot).
1) The density of cells per well
Totally agree with Anatoliy on the density. I had the same issue at the beginning of my experiments when some of my THP-1 cells detached. In my experiements, I was working on M0 and M1 phases. I ran some optimizations on the cell density but on 96-wells and 24-wells plates. Here's my parameters (perhaps you can adapt them to your 6-well plates):
RPMI-1640, 10% FBS, 1% L-Glutamine
96-wells - 4 x10^4 cells/ well in 100 μL
24-wells - 2.5 x10^5 cells/ well in 500 μL
PMA - 200 nM
PBS - use warm PBS to wash the cells gently
Incubation time: 48 hours
Use fresh PMA (prepared in small aliquots, do not re-thaw)
2) PMA stage to PMA(r) stage, with less % of FBS
One major change that I eventually did was prolonging the incubation time without PMA for 3 days, to help with the attachment. Check this paper: Article The Identification of Markers of Macrophage Differentiation ...
RPMI-1640, 1% FBS, 1% L-Glutamine
LPS - 1 μg/mL of E. coli 0111: B4
Incubation time: 72 hours (as according to the paper)
3) The optimal condition of THP-1 cells
Culturing THP-1 cells were quite tricky sometimes and similarly, I ran numerous times of optimisations and I cannot conclude consistent parameters for months. The other tip that I can share is I did not use or culture the THP-1 cells more than few cycles. What I eventually did was buying fresh THP-1 cells from ATCC and prepared many fresh stocks. From that point, I eventually was able to produce better and more consistent outcomes.
Hope it helps! I understand the feelings of optimising and working on the THP-1 cells for months but with so many inconsistencies!
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