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The use of a negative marker in conjunction with a positive marker is the most effective method of identifying fully reprogrammed colonies prior to expansion and selection. Somatic tissues like CD34+ blood cells, PBMCs, and human Fibroblasts express the surface marker CD44 at high levels. Expression of this marker has been demonstrated to be significantly down-regulated in fully reprogrammed cells. The combination of CD44 antibody staining with either SSEA4, Tra-1-60 or Tra-1-81 will more clearly distinguish fully reprogrammed colonies from partially reprogrammed colonies by the absence of CD44 and presence of self-renewal marker expression.
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