Human recombinant bdnf

Cultured cells were incubated for 24 h with 40 μM NTS (Calbiochem/Merck Millipore, Fontenay sous Bois, France) or 100 ng/ml human recombinant BDNF (Alomone labs, Jerusalem, Israel). Optimal concentrations of exogenous BDNF and NTS were determined previously.^{14 (link), 21 (link)} The non-peptide NTSR antagonist SR142948A (Tocris Biosciences, Bristol, UK) was used at 67 nM,^{56 (link)} PTX (Sigma-Aldrich, St Louis, MO, USA) at 200 ng/ml^{35 (link), 57 (link)}; TrkB inhibitors were K252a (Alomone Labs) at 100 nM^{58 (link), 59 (link)} or ANA12 (Tocris Biosciences) at 100 μM.^{60 (link)}

Cultured cells were incubated with 100 ng/mL human recombinant CXCL12^{20 (link)} (BioLegend, San Diego, CA, USA), 2 ng/mL human recombinant BAFF (BioLegend), and 25 ng/mL human recombinant APRIL (Peprotech, Neuilly-Sur-Seine, France) (these concentrations are comparable with the respective physiological levels in plasma from CLL patients^{21 (link),22 (link)}) or 100 ng/mL human recombinant BDNF (Alomone Labs, Jerusalem, Israel), as described previously^{12 (link),23 (link)}. BAFF was neutralized using a goat anti-BAFF polyclonal antibody (20 ng/mL; #AF124, R&D Systems, Minneapolis, MN, USA) and APRIL was neutralized using a mouse anti-APRIL monoclonal antibody (1 μg/mL; #MAB5860, R&D Systems)^{10 (link)}. CXCR4 was inhibited using AMD3100 (0.5 μg/ml; Merck Millipore, Fontenay sous Bois, France) to reduce the CXCR4-positive cell population markedly^{24 (link)}. BDNF was neutralized using an anti-BDNF mouse monoclonal antibody (200 ng/mL; #GF35L, Merck Millipore).