We are trying to quantify cholesterol in a macrophage, and I wonder what is the minimum number of cells we can use? Thank you.
Dont know if it helps, but for neutrophils I used 0.5-1*10^7 cells to quantify cholesterol.
Thanks for this information. Unfortunately, most if not all enzymatic assays for quantifying cholesterol do not work properly with macrophages; thus, GC is a good choice. In the past, we have used an HPLC approach which has been published as follows:
Cullen P, Tegelkamp K, Fobker M, Kannenberg F, Assmann G. Measuring cholesterol in macrophages: comparison of high-performance liquid chromatography and gas-liquid chromatography with enzymatic fluorometry. Anal Biochem 1997; 251(1):39-44.
Using this approach for quantifying cholesterol and cholesteryl esters in primary human macrophages, we used 75 cm2 culture flasks each containing 1 x 10^7 cells. At present, we are using GC-MS for quantifyin cholesterol and in parallel some of ist metabolic precursors. Using this approach, we obtain quite reliable result using 150 cm2 culture flasks each containing 2 x 10^7 cells. We also tried to use 1 x 10^7 cells in 75 cm2 culture flask. We obtained results with much higher variation. I hope that this information is valuable. All the best and please keep me updated.
Hi Stefan, could you tell me in concentration how much cholesterol (either total and/or free/esterified) when you used, let's say 10 million macrophages? I am asking this because then I will adjust the number of cells considering the sensitivity of our machine?
I did obtain 15 million macrophages, but I would rather measure it in triplicate than one measurement?? thanks in advance.
aicha