After washing, the cells were cultured in dishes covered with a s

After washing, the cells were cultured in dishes covered with a solution containing poly-L-lysine (200 μg/ml, Sigma Chemical, St. Louis, MO, USA), in DMEM containing 10% FCS, 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), 2 μM forskolin (Calbiochem, La Jolla, CA, USA), and 20 μg/ml bovine pituitary extract (Biomedical Technologies, Stoughton, MA, USA). After the first passage, SCs were further selected

from fibroblasts by using an anti-mouse Thy 1.1 antibody (undiluted hybridoma culture supernatant, American Tissue Culture Collection, Manassas, VA, USA) and rabbit complement (Sigma). This resulted in approximately 97 – 99% pure SC cultures as assessed by S100-β (DAKO, Carpinteria, CA, USA) immunoreactivity. SC-enriched cultures were maintained in a humidified air/CO2 (95%/5%) atmosphere at 37°C. Because a limited amount of primary

SCs was available, pilot experiments learn more were performed with the ST88-14 tumor cell line (Schwannoma cells). The ST88-14 cells, isolated from a patient with neurofibromatosis type 1 [24], were kindly donated by J.A. Flechter (Dana-Farber Cancer Sorafenib in vivo Institute, Boston, MA, USA). For inclusion in the present study, the cells were grown in RPMI 1640 medium supplemented with 5% FCS, 1 mM glutamine, 1000 U/ml penicillin, and 50 μg/ml streptomycin. All chemicals were from Sigma. The cells, plated in culture dishes or on cover slips in 24-well plates (Falcon, Franklin Lakes, NJ, USA), were maintained in a humidified air/CO2 SSR128129E (95%/5%) atmosphere at 37°C for 24 h. Phenotypic identification

of SCs The SCs cultures, both ST88-14 cells and Schwann cell primary cultures, were treated with PBS + 0.3% Triton X-100 (Sigma) and blocked with 10% normal goat serum (NGS). For phenotypic identification of SCs, the cultures were incubated with mouse monoclonal antibody anti-S100-β (Sigma), a Schwann cell marker [25]. After reaction with the primary antibodies of interest, cells were incubated with goat anti-rabbit IgG and/or goat anti-mouse IgG secondary antibodies. Soon after, the cells were washed in PBS pH 7.4 and mounted with N-propylgallate in PBS-glycerol and coverslipped. Expression of MR and uptake of a mannosylated neoglycoprotein by SCs SCs were tested for the expression of MR by labeling with a polyclonal antibody, produced in rabbits, directed against a C-terminal peptide of murine MR (anti-cMR, 1/100), kindly donated by Dr. Anne Régnier-Vigouroux [19]. A cytochemistry assay with 50 μg/ml of the neoglycoprotein mannosyl/bovine serum albumin-FITC-conjugated (man/BSA-FITC, Sigma) diluted in Ringer solution containing 5 mM CaCl2 and 1% BSA at 37°C for 1 h was performed in order to confirm the internalization pattern in SCs. Both expression and functional analyses (MR-mediated endocytosis) of the MR in SCs were performed as previously described by us in detail [20,7]. Interaction assay of S. pneumoniae and SCs Strain S.

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