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), a 40kDa transmembrane glycoprotein, is overexpressed in many solid tumors, while it shows variable but generally low levels in the normal epithelium.
Table 1: Details of PCR and RFLP procedures and expected products for hOGGI and APE1 genes Gene Primers PCR conditions PCR Forward/Reverse Product hOGGI 5'-ctg ttc agt 95[degrees]C- 5 m, 247 bp (C1245G) gcc gac ctg cgc 35 cycles of cga-3' 95[degrees]C-30 s, 64[degrees]C-30 s, Ser326Cys 5'-atc ttg ttg 72[degrees]C-30 s, cd326
tgc aaa ctg 72[degrees]C-5 m Exon7 ac-3' APE1 5'-ctg ttt cat ttc 95[degrees]C-5 m, 164 bp tat agg cta-3' 35 cycles of (T2197G) 5'-agg aac ttg 95[degrees]C-20 s, cga aag gct 55[degrees]C-20 s, Asp148Glu tc-3' 72[degrees]C-20 s, cd 148 72[degrees]C-5 m Exon5 Gene Restriction Restriction products enzyme hOGGI 2U of MbolI Ser/Ser: 224bp, 23bp (C1245G) Ser/Cys: 247bp, Ser326Cys 224bp, 23bp cd326
Cys/Cys: 247bp Exon7 APE1 1U of Bfal Asp/Asp: 144 bp.
Expression of surface molecules such as CD29, CD44, CD73, CD90, CD105, CD166, and human leukocyte antigen- (HLA-) ABC was positive in both donor- and RR-MS patient-derived hPDLSCs at P2 and P15 passages (Figure 1(a)), while that of CD14, CD31, CD34, CD45, CD326
, and HLA-DR was negative (data not shown).
In order to find putative impurities in the cell culture, cells were incubated in the dark for 30 minutes with fluorescein isothiocyanate- (FITC-) conjugated monoclonal antibodies against CD31(PECAM-1) (number F8402, Sigma-Aldrich), a marker of endothelial cells, and CD326
(EpCAM) (number 342203, BioLegend), a marker of epithelial cells.
confirmed that spheres from clinical specimens showed self-renewal, clonogenic, and tumorigenic potential and identified that they had lower expression levels of CD326
, CD24, CD44, and higher ABCG2 .