I did an experiment couple of days ago, the aim of the experiment I did was "differentiate U937 monocytic cells into macrophages usingPhorbol 12-myristate 13-acetate (PMA) and then quantify TNF-α production following LPS stimulation using a sandwich ELISA."
I already got my results (Attached) for differentiating U937 monocytic cells into macrophages using Phorbol 12-myristate 13-acetate part and quantify TNF-α production following LPS stimulation using a sandwich ELISA part.
However, I am not sure about what I should write about my results.
(Graphs attached).
Here is method of the experiment if you would like to read:
U937 cells were sub-cultured in RF-10 medium (RPMI 1640 medium supplemented with, 10 % foetal calf serum (FCS),100 units/ml penicillin, 100 𝜇g/ml streptomycin and 2 mM glutamine) in T25 tissue culture flasks. Cells were counted using a haemocytometer and resuspended at 0.5 × 106cell/ml in RF-10 medium containing 100ng/ml PMA and 200µl added to each well of a 96-well microtitre plate and incubated for 72 hours at 37°𝐶 to induce differentiation. Following incubation cells were pelleted by centrifugation and the supernatant discarded. 200𝜇l of RF-10 media containing a range of 0-2000 ng/ul of LPS was added to each well and post-incubated for 92 hours. Cells were then pelleted by centrifugation and 180𝜇l of supernatant was transferred to corresponding wells in a clean 96 well microtitre plate and stored at 4°𝐶.
100𝜇l of anti-TNF𝛼antibody was added to every well of an ELISA plate, and incubated overnight at 4°𝐶 in humidified conditions. The antibody was then replaced with 100𝜇l of ELISA diluent (PBS Tween containing 3% milk protein) and incubated for 1hour. The plate was washed and 50𝜇l of ELISA diluent added to all wells. A standard curve of TNF𝛼at 0-1000 pg/ml was prepared. 50𝜇l of culture supernatant from the microtiter plate was transferred into corresponding wells of the ELISA plate then the plate incubated for 20 minutes, washed and 100𝜇l of peroxidase conjugated secondary anti-human TNF𝛼added and incubated for a further 20 minutes. Excess conjugated antibody was removed and, following washing, 100𝜇l 0.4mg/ml OPD substrate + 0.01% H2O2 was added per well and incubated for 10 minutes at room temperature before the reaction was stopped using 25𝜇l of 12.5% sulphuric acid and absorbance read at 492nm using a multiscan-spectrophotometer.
Just some general comments... your ELISA standard curve needs some work. You will have issues repeating experiments if your standard curve looks like that. Also, I would use a 4-parameter graph for the curve fit.
Now... are you asking with the TNF goes down with higher LPS? LPS is cytotoxic at high concentrations... Also, 92 hours is quite long for LPS induced TNFa. We usually look around 24 hours. Of course, I guess it depends on what your goal is for the 92 hour time point.
Just some general comments... your ELISA standard curve needs some work. You will have issues repeating experiments if your standard curve looks like that. Also, I would use a 4-parameter graph for the curve fit.
Now... are you asking with the TNF goes down with higher LPS? LPS is cytotoxic at high concentrations... Also, 92 hours is quite long for LPS induced TNFa. We usually look around 24 hours. Of course, I guess it depends on what your goal is for the 92 hour time point.
I'd like to reiterate the above.
You should be able to get a might tighter standard curve. This is not only indicative of the pipetting you use for your entire protocol, but will severely impact your results. Also is there some reason your X-axis (LPS conc.) goes from zero, then highest to lowest?
I am also wondering on what basis you chose your LPS concentrations and time points?
Macrophages are highly sensitive to LPS. For example, we have used THP-1 derived macrophages via 48h PMA, 24h PMA-free. Exposure to 10ng/mL LPS fr 4 hours. ELISA consistently found 100-200pg/mL of TNFa. 1000ng/mL is a concentration used to stimulate much less responsive epithelial cells.
As above, dead cells cannot produce TNFa, and given its short half-life, any cytokine that was produced early on will have easily degraded by your end time point. You should definitely confirm your results and the above comment with a viability assay. LDH is quick and widely used, but there are other options.
First graph, your standard curve is not very well and second if you want to generate the formula for further calculations you have to use the semi-log or log way and then you can calculate your sample results.
Second graph, ... I think you used very high doses of LPS and killed the Mo (higher then 500 ng/ml). Usually the highest dose used for this in vitro issues in Mo is around 1000 ng/ml (mostly the optimal one is 100 ng/ml), so I would conclude that your cells with an higher conc. then 500 ng/ml are dead. In order to further elaborate this issue an MTT assay would be very helpful to check the viability.
Hello Nina,
As I work on the same experimental design, I can share some of my experiences.
1. 100ng/ml PMA is too cytotoxic. Adding LPS to it will even worsen the cell survival. I would suggest you standardize your experimental conditions and compound concentrations before you go in for experiments. 20ng/ml PMA works for me with over 95% of MDMs expressing CD14b at 72 hours. Please the check the survival of differentiated macrophages by checking the number of cells attached to the well before you pellet them and centrifuge.
2. You can scale up the experimental design to 24 well plates instead of 96 wells. I usually dont disturb the attached macrophages. Medium change should be done after every 72 hours. Shake the plate well and aspirate the unattached cells, wash with pre-warmed PBS, shake and aspirate again and finally add new pre-warmed medium. Now you can add LPS in the new medium. By this procedure, you can ensure that you are working with only live cells. And I am not sure if you disturb the attached cells they would re-attach again. Attachment has some significance with their function.
3. Use uncoated plates for macrophage culture. Coated plates have some effect on the transcriptome of macrophages (personal observation).
4. There is an inherent flaw with PMA. It differentiates monocytes only into "macrophage like cells" and never into "macrophages" (check the article in link: Article A Comparison of Differences in the Gene Expression Profiles ...
I wanted to use GM-CSF instead of PMA for differentiation and then use IFNg to polarize U937 into M1 macrophages before testing on them, but I never tried it. And I could not find literature which uses GM-CSF to differentiate U937 as everyone uses PMA for cell lines and GM-CSF for primary cells. Please let me know if you try it. I believe that GM-CSF differentiated cells would be a better experimental model than PMA differentiated U937.
5. ELISA would work only after you standardize all these conditions. So I am not commenting on your ELISA now.
Feel free to contact me for further details. Good luck with your experiment.
Best wishes,
Jeremiah
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