Hello,
I am currently working with 3T3 L1 cells and allowing them to differentiate into 24 well plates. After that, I would like to stimulate lipolysis with forskolin and sphingosine-1-phosphate and determine the glycerol release into the medium. Does anyone have any tips that I should consider for this? I would be very grateful for any help.
Lisa
Yoy should measure glycerol in the medium before and after treatment. Media contain a lot of free glycerol. There are kits for that measurement. Good luck.
Lipolysis can be measured by both Glycerol or Free Fatty acids in the culture media. The measurements can be done after 1 h of stimulation with Forskolin. Supplement media with 0.5mM IBMX.
Glycerol concentrations can be measured by using a glycerol assay kit (MAK117-1KT Sigma)
Hello Lisa Walz
Starve the cells for 2 hrs before treatment. You can supplement the Forskolin in KRH Buffer supplemented with 5mM Glucose and there is no need for FBS.
You can collect the supernatant at different time intervals like 0, 30, 60, and 120 minutes or 0,3,6,12 and 24 hrs.
After that, you can detect the Glycerol and NEFA in the collected supernatant.
You can collect the lysate after collecting the supernatant to normalize the content.
Thank You
Ref :- PMID- 28659738, 33758172
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays