1. Could someone recommend a culture media for human PBMC cultivation?
2. What concentration of LPS should I use to induce inflammation in cultured PBMC?
I found that concentrations between 100ng/ml up to 1µg/ml are used, is that the correct range?
We use RPMI and supplement it with 5% fetal bovine serum and 1% penicillin/streptomycin.
We both 100ng/ml and 1ug/ml and found they give the same response for IL-1beta/TNF-alpha. It may be better to use 100ng/ml since 1ug/ml is high.
We use RPMI and supplement it with 5% fetal bovine serum and 1% penicillin/streptomycin.
We both 100ng/ml and 1ug/ml and found they give the same response for IL-1beta/TNF-alpha. It may be better to use 100ng/ml since 1ug/ml is high.
I agree with Obada. We also use RPMI supplemented with 10%FBS, 1% p/s, and 1% L-glut. We stimulate with 100ng/mL and 1ug/mL as well and saw a response with IL-8. The 100ug/mL was sufficient to give a response as early as 3 hours. What time points are you looking at?
While I agree with various suggestions, I would like to mention that Oskan is talking about molarity (mol/ml or so) while the other folks give the mass concentration (g/ml).
I agree with all the previous answers. Serum might interfere with the LPS activation of the cells. An alternative is to heat-inactivate the serum.
Javier is right. If you use serum, it must be heat inactivated. For RPMI, serum is most certainly required to provide LPS binding protein, without it, TLR4 and CD14 cannot bind LPS. Be sure you use high-quality serum as endotoxin (LPS) is frequently present. Most suppliers will test for this and guarantee a maximum level in their more expensive line.
I did not decided about the time points yet. May be in the range between 3-12 hours?!
Could you give me some advices!
For a pilot experiment you could use anything from 4 to 24 hours according to your schedule. 6 hours is currently used in our protocols but this might mean a long day especially if you have another treatment after the LPS. I have seen papers where 24 h was used as well. Good luck!
The stimulation time will depend on the endpoint of your assay. If for example, you're measuring some cytokines, there are some papers reporting kinetics of production (like TNF-a, IL-8 etc) in response to LPS, 6h would be a good time. NF-kB translocation can come as early as 30 min. Inflammation is to broad, define how you want to assess it and then a good time point can be easily estimated.
Javier and Evan provided very good suggestions. For human PBMC RPMI supplemented with 10% of heat inactivated FBS, penstrep and L-glutamin is OK. LPS can be used at 10 ng/ml as well. Timing depends on read-out. Gene expression: early- 30 min-1h, intermediate - 3h, late after 6. You can also collect sups for ELISA or bead assay for cytokines after 1h. It all depends on the assays that you are planning to use. I would suggest to look at the literature, there are plenty of papers about LPS responses. Good luck.
I was also worked for the same and I found that when I use LPS 10ug/300ul the response was good as compared to when I used LPS 1ug/300ul RPMI media
We use RPMI 1640 with 5% HS - human serum without antibiotic, concentration of LPS - 1 μg/mL, after 24 h collect supernatants for ELISA IL-10, IL-6 , TNF-alfa and many other. We have good resulsts. Remember about control :). Good luck!
Oskan, you got some very good comments above. LPS is supplied from many sources, ideally you could test several concentrations (as mentioned above) once you decide which LPS you will use. It comes from different bacteria (e.g, E coli, Shigella, Salmonella), and different extraction/separation/purification methods. Timing depends on what your readout will be. If you need to look at mRNA, have to test between 30 min and no more than 12 h. If you'll test secreted cytokines, from 2 to 24, depending which cytokine is your target. We use RPMI with 10% FCS, or even better, defined medium with no serum (X-vivo 20). Preferable without antibiotics; these can mask a potential contamination and you want to keep your system totally clean. Good luck.
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