Metabolic characterization of a paused-like pluripotent state

Effects of DMSO on the Pluripotency of Cultured Mouse Embryonic Stem Cells (mESCs)

“…The enzyme was inactivated by dilution with fresh medium, and a small aliquot was used for Trypan Blue staining (Sigma-Aldrich) at a 1 : 1 ratio. Cell count was performed with a hemocytometer, and total number of cells per well was calculated [ 21 , 22 ]. Growth rate was then calculated using the mathematical formula—growth rate = (ln( x + 24h) − ln( x ))/24h, were x stands for the total number of cells in one time point and x + 24 h stands for the total number of cells counted 24 hours after x .…”

“…The immunocytochemistry protocol was performed as previously described [ 22 , 24 ]. In summary, cells were rinsed with warm PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 minutes at room temperature, rinsed three times with PBS, permeabilized with methanol at -20° C for 5 minutes, rinsed three more times with PBS, and blocked with a 5% BSA (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) PBS solution at room temperature.…”

“…Flow cytometry was performed to evaluate alterations in cell cycle profile and apoptosis/necrosis [ 22 , 23 , 25 ]. Samples were analyzed in a Becton Dickinson BD FACSCalibur cytometer, and 20,000 cells were gated per condition and evaluated with the Cell Quest Pro Acquisition software (BD Biosciences).…”

“…Evaluation of apoptosis and necrosis was performed using the Annexin V (Immunostep) and PI protocol [ 22 , 23 ] at the 24 and 48 hour time points. After harvesting, cells were centrifuged, washed once with PBS and 10 6 cells/ml, per condition, were resuspended in Annexin V binding buffer (10 mM Hepes/NaOH (pH 7.4) 140 mM NaCl, and 2.5 mM CaCl2), and incubated in the dark for 15 minutes at room temperature with Annexin V (50 μ L/ml) and 2.5 μ g/ml PI.…”

“…In this protocol, NZYol was added to the pellet of harvested cells, and after disruption with a vortex, chloroform (Sigma-Aldrich) was added. Subsequently, the mixture was centrifuged to separate a lower green phenol-chloroform phase with an interphase from a colourless upper aqueous phase containing the RNA, which was then collected and precipitated with 2-propanol at a 1 : 1 ratio [ 22 , 26 , 27 ]. Possible genomic DNA contamination was eliminated using the DNA-free kit (Ambion, Invitrogen).…”