Furthermore, harvested cell pellets were placed into vials containing 3% glutaraldehyde in 0.05 M phosphate buffer (pH 6.8) at 4°C and fixed for 48 h and then dehydrated in an ethanol series (30%, 50 %, 70, and 95%) for 20 min in eand alcohol dilution and finally with absolute alcohol for 45 min.
In vitro expanded, autologous preadipocytes have the potential for future therapeutic applications, since adipocytes, ATMs and ASCs are an abundant and easily harvested cell type.
After 3 days, we harvested cell pellets by centrifugation at 500 g for 15 minutes.
Experiments were performed using at least 3 individually harvested cell populations per group, and each sample was run in triplicate.
After 24 h incubation period, harvested cell suspension (10 μL) was added with equal volume of 0.4% trypan blue stain.
Following DNA transfections, cells were harvested, cell extracts prepared, and protein concentration determined by Bradford assay (BioRad, Hercules, CA).
Then, cell supernatants were harvested, cell debris was removed by centrifugation at 13.000× g, and samples were stored at −20 °C until further processing.
The fermentation was initiated by adding harvested cell suspension to reach a yeast concentration of 3 g dry weight L-1.
For enzymatic analysis of intracellular glycogen, 5 ml samples of respective cultures were harvested, cell extracts were prepared and glycogen content was determined with amyloglucosidase as described previously [ 8].
At the end of the incubation period, cell supernatants were harvested, cell debris was removed by centrifugation at 13,000× g, and samples were stored at −20 °C until further processing.
To ensure that both EB1 and EB3 are fully depleted in our time-lapse microscopy studies, we harvested cell extracts at the end of each time-lapse imaging session and quantified the extent of protein depletion using fluorescent immunoblotting.
