Following a 3-day subculture with glutamine or in glutamine-free medium with DMK (1.75 mM, SIGMA) and/or Nucleosides (Cytidine: 7.3 mg/L; Guanosine: 8.5 mg/L; Uridine: 7.3 mg/L; Adenosine: 8 mg/L; Thymidine: 2.4 mg/L; EmbryoMax, Millipore), HE cells were collected with StemPro Accutase Cell Dissociation Reagent and seeded onto OP9-DL1 stroma (80% confluent). The OP9-DL1 murine cell line was kindly provided by Dr. Ewa Sitnicka-Quinn (Lund University, Sweden) and its authentication was described in Renoux et al.41 (link). The protocol used to induce NK cell differentiation was described previously by Renoux et al.41 (link). Briefly, co-cultures were kept in OP9 medium, consisting of OptiMEM medium with Glutamax (Invitrogen) with 10% FCS, 1% penicillin–streptomycin solution (Thermo Fisher Scientific) and 1% 2-mercaptoethanol (Invitrogen) with SCF (10 ng/ml), FLT3-L (10 ng/ml), IL-2 (5 ng/ml), IL-7 (5 ng/ml, first 15 days only) and IL-15 (10 ng/ml). Cells were passaged onto new OP9-DL1 stroma every week. At day 35, cells were stained for human CD45 and CD56 markers and analyzed on a BD LSRFortessa.
Optimem medium with glutamax
Manufactured by Thermo Fisher Scientific
OptiMEM medium with Glutamax is a cell culture medium designed to support the growth and maintenance of a variety of cell types. It contains essential nutrients, vitamins, and the amino acid glutamine, which is a critical component for cell metabolism. This medium is intended for use in in-vitro cell culture applications.
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2 protocols using optimem medium with glutamax
1
Differentiation of HE cells to NK cells
2
Hematopoietic Differentiation on OP9-DL1
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Subculture day 3 HE cells cultured in the presence of UK5099 (10 µM) or DCA (3 mM), as indicated, were collected after a 2‐min incubation at 37°C with StemPro Accutase Cell Dissociation Reagent and seeded onto 80% confluent OP9‐DL1 stroma. Cells were cultured in OP9 medium (OptiMEM medium with Glutamax (Invitrogen) with 10% FCS, 1% penicillin–streptomycin solution (Thermo Fisher Scientific) and 1% 2‐mercaptoethanol (Invitrogen)) with SCF (10 ng/ml), FLT3‐L (10 ng/ml), IL‐2 (5 ng/ml), IL‐7 (5 ng/ml, first 15 days only), and IL‐15 (10 ng/ml) with passaging onto new OP9‐DL1 stroma every week, as described previously (Renoux et al, 2015 (link)). At day 35 of co‐culture, cells were analyzed on a BD LSRFortessa.
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