Some laboratories are interested detecting and counting the number of GFP-labeled adipocytes. The strongly green fluorescing cells are detected and counted (green outlined circles) while the weak and non-fluorescing adipocytes are not counted (red outline circles). Once the counting is complete, the Cellometer software reports the total number of counted cells in bright field and the number of cells that are GFP positive, the concentration, the mean cell diameter of each population, as well as the percent of GFP positive cells.
Selected Cellometer Application Literature Review
Stress stimulates production of catecholamines in rat adipocyte
Kvetnansky R, Ukropec J, Laukova M, Manz B1, Pacak K2, Vargovic P
Cellular and Molecular Neurobiology. 2012 July; 32(5): 801-813
Institute of Experimental Endocronology, Slovak Academy of Sciences, Bratislava, Slovak Republic
1LDN Labor Diagnostika Nord, Germany
2National Institute of Child Health and Human Development, NIH, Bethesda, USA
In adipose tissues, catecholamines (CAs) play a key role in the regulation of adipose tissue metabolism. The aim of this study was to investigate the effect of different stressors (emotional – immobilization and physical – cold) on CA production in adipocytes. Stromal vascular fraction (SVF) was isolated from mesenteric adipose tissue of rats during stress exposure. In this publication, researchers reported that stress induced CA production in adipocytes, thus suggesting a direct involvement of this novel CA system in the regulation of stress response. The larger overall importance of this research indicates that the stress response in mesenteric adipose tissue was associated with stress-induced adipogenic (fatty tissue production) program, providing a direct link between stress, obesity, and metabolic disease.
Mesenteric adipose tissue (200–300 mg) was collected from the rats. After collagenase I digestion (30 min, 37°C, with constant gentle agitation), floating fraction of adipocytes was subsequently washed 4-times with 1.5 ml of medium. Each washing step was followed by centrifugation. The pelleted cells were separated from the floating cell layer by washing and filtering. Floating cell suspension was filtered through 210 μm nylon mesh filter to remove any remaining cell clumps potentially harboring non-adipocyte cells. Fraction of centrifugation pelleted cells separated from adipocytes was considered to be the stromal fraction.
Because lipid laden adipocytes float to the top, researchers used this to separate the floating adipocytes from the non-floating cells. They then analyzed the non-floating cells before and after each washing step. Upon collection of the lipoaspirate, the floating fraction of adipocytes was collected and washed. (1st wash) The collected top fraction was washed and spun down. The bottom layer (here called: Infranatant) was analyzed for the presence of immune cell. (2nd wash) The top layer of adipocytes from the 1st wash was collected, washed, and spun down. Again, the bottom layer was analyzed for immune cells. This was done 4 times.
“The purity of adipocyte preparation was monitored by automated histomorphological analysis with Nexelcom, cellometer T4 plus (Nexcelom Bioscience, LLC)”