Assuming a cellular average for the relationship between density and changes in refractive index, the measured phase shift at each location inside of the cell is directly proportional to the amount of biomass at that location, resulting in a map of the distribution of mass for all nonaqueous components (dry mass) (12, 13, 14?and?15). In this study, we assume that the conversion factor between phase shift and mass, the inverse of the specific refractive index, is 5.56 pg/��m3, although <a href="http://www.selleckchem.com/products/byl719.html
">selleckchem</a> we note that this parameter only varies by ?��10% among the typical contents of a cell ( 12?and?13). Assuming the average contents of the cell/colony remain fairly constant over the measurement period, the specific value of this parameter will drop out of any measurements based on relative mass comparisons, such as the specific growth rate. Recent applications of quantitative phase microscopy using other imaging platforms include measurements of cell growth ( 14), cell death ( 16), membrane mechanics ( 17), individual organelles ( 18), and preliminary imaging of mouse PSCs ( 19). Prior work with LCI establishes it as a method for repeatable (<2% coefficient of variation) quantification of mass, mass accumulation rate, and mass distributions for large populations of cells on a single cell basis ( 13?and?20), but not for cells constrained within hPSC colonies. HSF1 human embryonic stem cells (hESCs; 46XY; UC-0001) were grown on feeder-free Matrigel (BD Biosciences, San Jose, CA) in defined cell culture media (StemPro SFM; Invitrogen, Carlsbad, CA) with <a href="http://www.selleckchem.com/products/MG132.html
">MG-132 in vitro</a> daily media changes, as described previously in Zhang et?al. (21?and?22). Differentiation with retinoic acid (RA) was induced by replacing bFGF with 10-��M RA (Acros Organics, Fair Lawn, NJ). Cell counting was performed using a Neubauer hemocytometer after Trypan-blue staining (Invitrogen). <a href="http://www.selleck.cn/products/Decitabine.html
">Decitabine</a> Embryoid body (EB) formation was performed as described previously in?Zhang et?al. (21). Briefly, hESCs were trypsinized to single cells and 106 cells were placed in one well of a AggreWell 400 plate (Stemcell Technologies, Vancouver, BC, Canada) according to the manufacturer��s instructions. The next day, EBs were harvested and grown in Iscove��s Modified Dulbecco��s medium supplemented with 20% fetal bovine serum for 10?days in a Corning (E. I. DuPont de Nemours, Wilmington, DC) CoStar ultra-low attachment six-well plate (Sigma-Aldrich, St. Louis, MO). Media was exchanged every 2�C3?days. An inverted light microscope was used to assess and count EBs. Total RNA was extracted from control or RA-exposed hESCs using Trizol reagent (Invitrogen) according to the manufacturer��s instructions.?cDNA was synthesized from total RNA using a Superscript III first-strand cDNA synthesis kit (Invitrogen). Real-time PCR was performed with a SYBR Green PCR kit (Diagenode, Denville, NJ) with denaturation at 94��C for 15 s, annealing at 60��C for 30 s, and extension at 72��C for 45?s over 40 cycles.