Schwann cell medium

Mouse Schwann cell primary cultures were obtained from ScienCell Research Laboratories™ (San Diego, CA, catalog #M1700) and were cultured according to the manufacturer’s instructions. Briefly, cells were cultured in Schwann cell medium, supplemented with 5% fetal bovine serum, 1% Schwann cell growth supplement and 1% penicillin/streptomycin (all from ScienCell Research Laboratories). After monolayer propagation in T-75 flasks at 37°C in a 5% CO2 humidified chamber, 5×10⁵ cells were seeded in 12-well plates and incubated with WT or V30M TTR, both at 5 μM in triplicates. In a different experiment Schwann cells were incubated with Cli-095 (InvivoGen, San Diego, CA, catalog #tlrl-cli95) and sRage which was isolated from bacteria inclusion bodies (21 (link)), antagonists for TLR4 and Rage respectively, before a second incubation with WT or V30M TTR. Twenty-four hours after stimulation, supernatants were collected and cell lysates were prepared in trizol (Invitrogen, Waltham, MA, catalog #15596026) and assayed for the expression of proinflammatory cytokines by RT-PCR. Unstimulated cells were also used as controls.

HEK293 (ATCC), MCF-10A (ATCC), primary Schwann (ScienCell), and immortalized human normal Schwann cells (Margaret Wallace, University of Florida)^{63 (link)} were authenticated using short tandem repeat profiling at MSKCC IGO and tested for mycoplasma using the PCR-based Universal Mycoplasma Detection kit (ATCC). HEK293 and immortalized Schwann cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MCF-10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml epidermal growth factor, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, and 1% penicillin/streptomycin. Primary Schwann cells were cultured in Schwann Cell Medium (ScienCell), consisting of basal medium, 5% FBS, 1% Schwann cell growth supplement, and 1% penicillin/streptomycin. All cell lines were maintained in a 5% CO₂ atmosphere at 37 °C.

Human Schwann cells (HSCs) were purchased from ScienCell Research Laboratories (catalogue no., 1700; Carlsbad, CA, USA), and cultured in Schwann Cell Medium (ScienCell, Cat. No.1701). For primary schwannoma cell culture, tumour tissues were cut to 2 mm³ in size and digested with 0.25% trypsin. The cells were collected and re-suspended in culture medium: DMEM (PAA, Coelbe, Germany), 2.5% Nu-Serum (BD), 0.5 µM forskolin (Sigma) and 10 nM β1-heregulin (Peprotech EC Ltd., UK). To establish merlin-knockdown cultures, short hairpin RNAs (shRNA) were synthesized, annealed, and inserted into a lentivirus vector, containing an independent open reading frame for green fluorescence protein (GFP). Lentiviral shRNA (Lv-shRNA) vectors were constructed using three different shRNA sequences against NF2/merlin expression including 5′-ACTTCAAAG ATACTGAC AT-3′ (sh1), 5′-TCTGGATA TTCTGCACAAT-3′ (sh2), and 5′-TTCGTGTTAATAAGCTGAT3′ (sh3). A nonsense shRNA was also constructed using the target sequence 5′-TTCTCCGAACGTGTCACGT-3′ (GeneChem, Shanghai, China). The lentivirus-mediated shRNAs were used to transfect schwannoma cells at a multiplicity of infection (MOI) of 20. To screen the target for the most effective viral transfection, the percentage of GFP-positive cells in the total cell numbers was evaluated under the fluorescence microscope at day 3 following transduction.