AFSCs were thawed, total RNA was extracted, and Oct-4 and Nanog gene expression was determined by RT-PCR in 2% agarose gel stained with ethidium bromide after freezing with different cryoprotectants. Human β-actin was used as endogenous control. MW: 100 bp molecular weight; A: 5% Me2SO nonprogrammed; B: 5% Me2SO, programmed; C: 10% Me2SO, nonprogrammed; D: 10% Me2SO, programmed; E: 5% Glycerol, nonprogrammed; F: 5% Glycerol, programmed; G: 10% Glycerol, nonprogrammed; H: 10% Glycerol, programmed; I: 30 mM Sucrose, nonprogrammed; J: 30 mM Sucrose, programmed; K: 60 mM Sucrose, nonprogrammed; L: 60 mM Sucrose, programmed; M: 60 mM Trehalose, nonprogrammed; N: 60 mM Trehalose, programmed; O: 100 mM Trehalose, nonprogrammed; P: 100 mM Trehalose; programmed; Q: positive control (Ntera-2.c1D1 cell line).