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Journal of Oral Science & Rehabilitation Volume 2 | Issue 2/2016 29 H u m a n i n d u c e d p l u r i p o t e n t s t e m c e l l s f r o m h u m a n o r a l m u c o s a Table 2 Antibodies used for flow cytometry and immuno- chemical staining of HOF-iPSCs. Table 2 S u r f a c e a n t i g e n a n a l y s i s Cells(5×105 )wereobtainedaftertreatmentwith 0.025% trypsin (Life Technologies). Cell surface antigen staining was performed in phos- phate-buffered saline (PBS) with 2% human serumalbumin(Mitsubishi-TanabePharma,Osa- ka, Japan). The cell suspension was incubated withthe antibodies listed in Table2for30min at 4°C. Murine anti-human antibodies were used at the recommended concentrations. Primary antibodiesandisotypecontrolsarelistedinTable 2. The stained cells were analyzed with FACSAr- ia II (Becton Dickinson, Franklin Lakes, N.J., U.S.) and the data were analyzed using the FlowJo software (Tree Star, Ashland, Ore., U.S.). I m m u n o c y t o c h e m i s t r y Forfixedstainingofdifferentiation-specificmark- ers, cellswerefixedfor30min in 4% paraformal- dehyde at 4°C, followed by washing in PBS. The cells were then permeabilized for 15min with 2% bovineserumalbuminand0.1%TritonX-100(Sig- ma-Aldrich) and incubated overnight at 4°Cwith the primary antibodies diluted in PBS containing 2% bovine serum albumin. The cells were then washedandincubatedfor1hwiththeappropriate fluorescence-conjugated secondary antibodies. Primary antibodies and secondary antibodies are listed in Table 2. The staining images were ac- quired with a ZOE Fluorescent Cell Imager (Bio- Rad Laboratories, Hercules, Calif., U.S.). I n v i v o d i f f e r e n t i a t i o n ( t e r a t o m a f o r m a t i o n ) NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ mice (Jackson Laboratory, Bar Harbor, Maine, U.S.) were anes- thetized and iPSCs (1×106 ) were transplanted underthe epidermalspace ofthe neck.Two hun- dred microliters of saline was injected into a second epidermal space as a negative control. Mice were euthanized 12 weeks later and tera- toma samples were collected and subjected to histological analysis. Teratomas were processed according to standard paraffin embedding and hematoxylin and eosin staining procedures by the Business Support Center for Biomedical Re- search Activities (Kobe, Japan). Antibodies Supplier Cat. No Dilution OCT3/4 Santa Cruz Biotechnology, Dallas, Texas, U.S. SC5279 1/100 NANOG Cell Signaling Technology, Danvers, Mass., U.S. 3680S 1/100 SSEA-3 Abcam, Cambridge, Mass., U.S. ab16286 1/100 SSEA-4 Millipore, Billerica, Mass., U.S. MAB4360 1/100 TRA-1-60 Millipore, Billerica, Mass., U.S. MAB4304 1/100 TRA-1-81 Millipore, Billerica, Mass., U.S. MAB4381 1/100 DAPI Invitrogen, Carlsbad, Calif., U.S. D1306 5μg/mL Alexa Fluor 594 mouse Invitrogen, Carlsbad, Calif., U.S. A11062 1/500 Alexa Fluor 594 rat Invitrogen, Carlsbad, Calif., U.S. A21211 1/500 Volume 2 | Issue 2/201629