I have bought Sigma Isoproterenol Hydrochloride (I6504). I have dissolved it in KRBH buffer (pH-7.4) to make it 50 mM stock and stored @ -20. I used 10 micromolar conc. to treat adipocytes for 1-2 hr. But I am not getting any desired effect. One protocol used 1 mM HCL to dissolve isoproterenol.
http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PL00000265.pdf?pid=PL00000265
What I have done wrong? Please suggest.
Hi,
Have you tried 4h of stimulation? I am afraid that 1-2h may not be enough to have detectable levels of lipolysis. How have you measured lipolysis, by Glycerol or NEFA levels?
I dissolved Isoproterenol Hydrochloride in H2O ant it worked perfectly...see Rodrigues et al (2013) Alpha-MSH signalling via melanocortin 5 receptor promotes lipolysis and impairs re-esterification in adipocytes. BBA, 1831(7):1267-75.
Good luck.
Adriana
Hey,
I think that 3 hours is enought to induce lipolyse. Have a look to my paper. It may helps you.
https://www.researchgate.net/publication/259985556_Efficient_in_vitro_adipocyte_model_of_long-term_lipolysis_A_tool_to_study_the_behavior_of_lipophilic_compounds
We also made a time-lapse to illustrate the progression of lipolysis. The video in available online.
Good luck!
Article Efficient in vitro adipocyte model of long-term lipolysis: A...
hello, I agree that maybe you need more time to find a lipolytic effect
Hi, I dissolve the isoproterenol in H2O and can induce lipolysis after a 2h treatment. You could try a dose response to see if the dose is too low (which I don't think it is). Alternatively you can try a 4 h incubation and take an aliquot at 2h as well. If that does not work then there may be a problem with your cells. What size plates have you seeded them in?
It is best not to strictly follow a particular method; so try a modification because of the peculiarity of your substrate and preparation procedures. And also pay attention to purity of reagents, especially the buffer. We have gone through the protocol successfully based on slight modifications of existing methods. Good luck in your work.
Hi Thomas, Thank you for your answer. I have seeded in 12 well plate, but efficiency of transformation to adipocytes is less in 12 well plate than 6 well plate. That's what I have observed. Should I do in 12 well plate.
Hi Adriana,
I didn't try 4 hour stimulation. I will do it next time. I have measured glycerol levels using Cayman Glycerol Cell based assay kit.
I think you should serum starved cells an O.N. (at least 6h) prior stimulation to get higher rates of stimulation.
Hi All,
I have tried everthing as you suggested. I serum starved it also for min 6 hours but still didn't get any stimulus with Isoproterenol. However, IBMX has given me good stimulation. I am using Isoproterenol from Sigma (Cat No- I6504). I am afraid whether I am using correct Isoproterenol or not ?
Hi again Gautam,
That's unfortunate! Are your cells lipid loaded prior to treating them? That is the exact same isoproterenol that I use and I get stimulation in my cells.
Hi Thomas,
Nice to hear from you. Lipid loaded means whether they accumulate enough triglycerides or not?? I allow them to fully differentiate to become mature adipocytes. Almost 80-90% of them contains lipid droplets. I dont know what is happening Thomas. How do you prepare your Isoproterenol??
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