The triple deficient pro B cell line (Rag2−/−, λ5−/−, SLP65−/−) (TKO, Meixlsperger et al., 2007 (link)) is a kind gift of Hassan Jumaa. To generate the TKO-MD cells, TKO cells were retrovirally co-transfected with vectors encoding the λ1 light chain, B1-8 μm and δm HC carrying selection markers for puromycin and complemented YFP (Köhler et al., 2008 (link); Infantino et al., 2010 (link)). The resulting cells were stained with 1NIP-peptide-DyLight649 (custom synthesized from Biosyntan GmbH, Berlin, Germany) for surface NIP-specific BCR and sorted for cYFP+ and Dylight649+ on BD (San Jose, CA) Influx FACS sorter.
To transfect TKO cells, supernatants containing viral particles were collected from transfected Phoenix retrovirus packaging cells 2 days after plasmid transfection and used as described before (Duhren-von Minden et al., 2012 (link)).
Both the TKO cells and the TKO-MD cells were cultured in Iscove's medium (Biochrom, Berlin, Germany) supplemented with 10 mM L-glutamine, 100 unit/ml penicillin/streptomycin (all Gibco, Life Technologies, Darmstadt, Germany), 50 μM β-mercaptoethanol (Sigma-Aldrich, Munich, Germany), 10% FCS (PAN Biotech, Aidenbach, Germany) and supernatant of cultured J558L mouse plasmacytoma cells stably transfected with a murine IL-7 expression vector.
To transfect TKO cells, supernatants containing viral particles were collected from transfected Phoenix retrovirus packaging cells 2 days after plasmid transfection and used as described before (Duhren-von Minden et al., 2012 (link)).
Both the TKO cells and the TKO-MD cells were cultured in Iscove's medium (Biochrom, Berlin, Germany) supplemented with 10 mM L-glutamine, 100 unit/ml penicillin/streptomycin (all Gibco, Life Technologies, Darmstadt, Germany), 50 μM β-mercaptoethanol (Sigma-Aldrich, Munich, Germany), 10% FCS (PAN Biotech, Aidenbach, Germany) and supernatant of cultured J558L mouse plasmacytoma cells stably transfected with a murine IL-7 expression vector.
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