Hello all,
I am searching for a way to measure the activity of CPT1 and/or FAO in mouse hematopoietic stem cells, which means I have very little material available to do so.
Any suggestions?
Thanks a lot
Dear Claire!
Could You look at this article, plase:
https://www.nature.com/articles/s41598-020-58334-7
Cells
U-937, MCF-7, T47D, and MDA-MB-468 cell lines were obtained originally from the American Type Culture Collection (ATCC) (Manassas, VA). These cells were cultured in RPMI 1640 (2 g/l glucose, for U-937 and T47D) or DMEM (1 g/l glucose, for MCF-7 and MDA-MB-468) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.
FAO measurement
The cellular FAO activity was determined by quantifying the conversion of 3H palmitic acid to 3H2O over the labeling time. Cells were seeded in 12-well plates and when appropriate, treated with FAO inhibitors or stimulators. For FAO assays in complete medium with 10% FBS, [9,10-3H(N)]-palmitic acid [0.5 µCi (~9.3 pmol)] in 15 µl carrier solution (sodium palmitate 1 mM/BSA 0.17 mM/NaCl 150 mM)76 was added to each well (final concentration of added palmitate was ~15 µM, in addition to free fatty acids and other lipids present in FBS). For FAO assays in Krebs buffer or culture medium without FBS, the volume of carrier solution was increased to 50 µl, making a final concentration of palmitate at ~50 µM. After labeling, culture medium (0.5 ml out of 1 ml) was collected into a 15-ml Falcon polypropylene conical tube (#352096, BD Biosciences, San Jose, CA), and mixed with 100 µl of 1.2 N HCl to terminate all biological reactions. The 15-ml Falcon tube has a longer screw top to ensure tight closure than similar products of other brands. A 0.5-ml microcentrifuge tube containing 0.25 ml of sterile distilled water was uncapped and inserted into the 15-ml tube with forceps. Precautions were taken to prevent direct contact between the medium and water phases. The 15-ml tubes were tightly capped to allow diffusion between two liquid phases at room temperature for 3 days unless otherwise indicated.
Figure 1
Alexandr Chernov Thank you for your answer. I have indeed already looked at this paper and the technique they are using might be worth testing.