Journal: Scientific Reports
Article Title: Natural killer cells impede the engraftment of cardiomyocytes derived from induced pluripotent stem cells in syngeneic mouse model
doi: 10.1038/s41598-019-47134-3
Figure Lengend Snippet: Expression analysis of NKC ligands on iPSC-CMs with or without IFN-γ treatment and expression of NKC-activating receptors on purified NKCs before coculture. ( A ) Flow cytometric detection of MHC class I molecule expression on C57BL/6 splenocytes using antibodies against H2Db, H2Kb, Qa1-b, and Qa2, or isotype-matched control antibodies. ( B ) Flow cytometric detection of MHC class I molecule expression on iPSC-CMs with or without IFN-γ treatment. The iPSC-CMs were stained with antibodies against H2Db, H2Kb, Qa1-b, and Qa2, or isotype-matched control antibodies. ( C ) Flow cytometric detection of NKC-activating ligands on iPSC-CMs at day 16. The iPSC-CMs were stained with antibodies against MULT1, H-60, RAE-1, CD112, and CD155, or isotype-matched control antibodies. ( D ) Flow cytometric detection of MHC class II (I-A/I-E) molecules on iPSC-CMs with or without IFN-γ treatment, stained with anti-I-A/I-E, CD40, CD80, and CD86 antibodies or isotype-matched control antibodies. ( E ) mRNA expression of CIITA and NLRC5 in iPSC-CMs with or without IFN-γ treatment, as measured by real-time PCR. The results are expressed relative to those of C57BL/6 splenocytes as means ± SD (n = 3, respectively); *p < 0.05. ( F ) Flow cytometric detection of NK1.1 and CD3 expression on cultured NKCs purified from C57BL/6 splenocytes, stained with antibodies against NK1.1 and CD3, or isotype-matched control antibodies. ( G ) Flow cytometric detection of NKC-activating receptors on cultured NKCs purified from C57BL/6 splenocytes, stained with antibodies against CD226 and NKG2D, or isotype-matched control antibodies.
Article Snippet: Cultured NKCs were washed and incubated with blocking antibodies against DNAM-1 (CD226) (Thermo Fisher Scientific) and NKG2D (BioLegend) for 90 min at room temperature.
Techniques: Expressing, Purification, Control, Staining, Real-time Polymerase Chain Reaction, Cell Culture
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