Iscove s medium

All mouse housing, breeding, and surgical procedures were approved by the governmental institutions of Baden-Württemberg (Regierungspräsidium Tübingen). BM cells from WT (n = 7, female), Il7rα^Δ (n = 7, female), Il7rα^fl/fl (n = 7, female), Cxcr4^fl/fl (n = 3, female) and FoxO1^fl/fl (n = 3, female) mice were collected and retrovirally transformed with either an empty pMIG vector or with a pMIG vector expressing BCR-ABL1. Unless mentioned otherwise, cells were cultured for 3–7 days in Iscove’s medium (Biochrom AG) containing 10% heat-inactivated FCS (Sigma-Aldrich), 2 mM l-glutamine, 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco), and 50 µM 2-mercaptoethanol. The medium was supplemented in excess with the supernatant of J558L plasmacytoma cells stably transfected with a vector encoding murine IL7. Transformed cells were selected by IL7 withdrawal and kept in optimum conditions^{38 (link)}. Retroviral vectors containing either constitutively active STAT5 (STAT5-CA)^{34 (link)} or an empty vector (EV) were used to transduce BCR-ABL1-transformed cells and sorted cells were used then for western blot or flow cytometry analysis. 1–2 µM 4-hydroxy Tam (Sigma-Aldrich) was used to induce deletion on plasmids expressing Tam-inducible form of Cre (Cre-ER^T2)^{14 (link)}. All cells were tested and found free from mycoplasma.

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.

The murine cell line, 6606PDA and 7265PDA were a gift of Prof. Tuveson (University of Cambridge, UK) and were grown in DMEM high glucose medium (Biochrom GmbH, Berlin, Germany) as previously described [19 (link), 20 (link)]. The human MIA PaCa-2 cells were ordered from ATCC (LGC Standards GmbH, Wesel, Germany). The PSCs were isolated from the pancreas of C57BL/6J mice by collagenase digestion of the organ and by Nycodenz density gradient centrifugation as previously described [37 (link)]. These cells were expanded in Iscove's-medium (Biochrom GmbH, Berlin, Germany) supplemented with 17% fetal calf serum (FCS), 1% non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin for one to two weeks. All experiments were performed with passaging the cells no more than 2 times.

Ramos cells were cultivated in RPMI (Gibco) while Phoenix and mature splenic B cells were cultured in Iscove's medium (Biochrom AG). The media were supplemented with 5% heat‐inactivated FCS (Sigma), 2 mM l‐glutamine (Gibco), 100 U/ml penicillin/streptomycin (Gibco), and 50 μM β‐mercaptoethanol, respectively. For survival assessment of anergic B cells, 2.5 μg/ml lipopolysaccharides (LPS, Sigma) and/or 10 ng/ml murine IL‐4 (immunotools) were added to the medium.

TKO cells26 (link) and Phoenix cells (ATCC CRL-3214) were cultured in Iscove's medium (Biochrom) containing 10 mM L-glutamine (Gibco), 10% heat-inactivated fetal bovine serum (PAN-Biotech) and 100 U ml⁻¹ penicillin/streptomycin (Gibco). For culture of TKO cells, the medium was supplemented with 50 μM β-mercaptoethanol (Gibco) and supernatant of J558L mouse plasmacytoma cells stably transfected with a vector encoding murine IL-7 (a kind gift from T Rolink to H Jumaa).

The CD34+ cells were cultured using the 3-stage erythroid culture system. During the first 8 days the cells were maintained in Basic medium which was Iscove’s medium (Biochrom) containing 3% (v/v) human AB serum (Sigma-Aldrich), 10% fetal calf serum (Hyclone, Fisher Scientific, Ltd), 10 μg/ml insulin (Sigma-Aldrich), 3 U/ml heparin (Sigma-Aldrich), 3 U/ml EPO (Roche), 200 μg/ml transferrin (R&D Systems) and 1 U/ml penicillin/streptomycin (Sigma-Aldrich) supplemented with 10 ng/ml SCF (R&D Systems) and 1 ng/ml IL-3 (R&D Systems) (primary medium). IL-3 and SCF were withdrawn from the medium on day 8 (secondary medium) and 11 (tertiary medium), respectively. In addition, extra transferrin was added to the medium to the final concentration of 500 μg/ml from day 11 onward. The cells were counted and medium was added every other day. The cultured cells were maintained at 37 °C, 5% CO₂ throughout the culture period. At indicated time points, aliquots of cells were collected for morphological analysis using cytospin and Leishman staining. Two sample equal variance t-test was carried out to determine the statistic significances of cell numbers and cell types.

Primary B cell precursors cells were isolated from the bone marrow of wild type mice, the SLP-65^-/- pre-B cell lines Dec and Oct have already been described [30 (link)]. Pre-B cells were cultured in Iscove’s medium (Biochrom) containing 10% fetal calf serum (Vitromex), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin (Biochrom) and 50 μM 2-mercaptoethanol (Gibco) supplemented with excessive IL-7 obtained from the supernatant of stably transfected J558L cells. Schneider S2 cells (a generous gift from Dr. K. Karjalainen, commercially available as CRL-1963 through ATCC) were grown in Schneider’s Drosophila medium (Life Technologies) supplemented with 10% fetal calf serum at 27°C without CO₂ [4 (link)].