Robosep automaton

BM cells removed from tibiae and femurs of 8–12-week-old C57BL/6J mice were incubated in RPMI-1640 medium (PAA) supplemented with 10% (vol/vol) fetal calf serum and 1% antibiotics (penicillin and streptomycin) for 18 h with 1 μg ml⁻¹ of the oligodeoxynucleotides CpG 1,585 (CpG-A; InvivoGen), CpG 1,668 (CpG-B; Eurogentec, Angers, France) or with the respective agonists of TLR-1–9, supplied in a commercial kit (InvivoGen, Toulouse, France), including: Pam3CSK4 (0.5 μg ml⁻¹), FSLI (1 μg ml⁻¹), HKLM (2 × 10⁶ cells ml⁻¹), Poly I:C (HMW; 5 μg ml⁻¹), Poly I:C (LMW; 5 μg ml⁻¹), LPS-EK standard (1 μg ml⁻¹), FLA- ST standard (1 μg ml⁻¹) and ssRNA40/LyoVec (2 μg ml⁻¹) as well as CpG 1,826 (CpG-B; 1 μg ml⁻¹).
c-kit⁺ BM cells were sorted by immuno-magnetic separation using a RoboSep automaton (StemCell Technologies, Grenoble, France). Sorted cells were further stained with appropriate fluorochrome-conjugated mAbs against Sca-1, B220, PDCA-1, IgM and electronically sorted as a small-size, c-kit^lowSca-1^lowB220⁺PDCA-1⁻IgM⁻ cell subset using a FACS-Aria I (BD Biosciences, Le Pont de Claix, France).

Bone marrow (BM) cells removed from tibiae, femurs and hips of 8- to 14-week-old C57BL/6J mice were incubated in low endotoxin RPMI-1640 medium (Fisher Scientific, Illkirch, France) supplemented with 10% (vol/vol) FCS and 1% antibiotics (penicillin and streptomycin) for 18 h with 1 μg/ml of the oligodeoxynucleotide CpG 1668 (CpG-B) (Eurogentec, Angers, France) or with the respective agonists of TLR1-9, supplied in a commercial kit (InvivoGen, Toulouse, France), including: Pam3CSK4 (0.5 μg/ml), FSLI (1 μg/ml), HKLM (2 × 10⁶ cells/ml), Poly I:C (HMW) (5 μg/ml), Poly I:C (LMW) (5 μg/ml), LPS-EK standard (1 μg/ml), FLA-ST standard (1 μg/ml), ssRNA40/LyoVec (2 μg/ml) as well as CpG 1826 (CpG-B) (1 μg/ml). c-kit⁺ BM cells were sorted by immuno-magnetic separation using a RoboSep automaton (StemCell Technologies, Grenoble, France). Sorted cells were further stained with appropriate fluorochrome-conjugated mAbs against Sca-1, B220 (BD Biosciences, Le Pont de Claix, France), PDCA-1 (eBioscience, Paris, France or BioLegend, London, UK) and electronically sorted as a c-kit⁺Sca-1⁺B220⁺PDCA-1⁺ cell subset using a FACS-Aria IIIu (BD Biosciences).

CpG-proB cells were isolated from C57BL/6J BM cell cultures activated with 1 μM CpG-1668 (CpG-B) (Eurogentec, Angers, France) for 17h in low endotoxin-RPMI medium (Fisher Scientific, Illkirch, France) supplemented with 10% (vol/vol) FCS and 1% antibiotics (penicillin and streptomycin). c-kit⁺ cells were magnetically sorted using the Robosep automaton (StemCell Technologies, Grenoble, France) and thereafter stained with appropriately labeled mAbs and sorted by flow cytometry on a BD FACS Aria IIIu cell-sorter as c-kit⁺Sca-1⁺B220⁺PDCA-1^-IgM^- cells. Electronically sorted B-cell progenitors were cultured on plates at 20,000 cells/ml over OP-9 stromal cells in OPTIMEM medium (Gibco) supplemented with 10% FCS, 1% antibiotics, 0.1% β-mercaptoethanol and 20 ng/ml Flt3L, SCF (Immunotools, Frisoythe, Germany) and IL-7 (Peprotech France, Neuilly-sur-Seine, France), achieving on average a 10-fold expansion of sorted CpG-proBs over 6 days. Expanded CpG-proBs were further stained and electronically sorted as c-kit^low/- Sca-1⁺B220⁺PDCA-1^-IgM^- cells, routinely assessed as >95% pure, before i.v. injection through the retro-orbital sinus.