Trypsin edta 1

Brachial DRGs were dissected and collected in Leibovit’z L-15 medium (Life technologies) on ice. Then the DRGs were incubated in 0.05% trypsin-EDTA (1×) (Life technologies) for 5, 7, 10 and 15 min for DRGs from E9.5, E10.5, E11.5 and E12.5, respectively, at 37 °C, in a thermomixer comfort (Eppendorf) at 700 RPM. After spinning down the samples at 100 RCF for 5 min, the supernatant was removed and replaced by Leibovit’z L-15 medium (Life technologies). DRGs were physically triturated using two different sizes of pipettes previously coated with 0.2% bovine serum albumin until the solution homogenized. The cell suspension was then filtered through a 70-μm cell strainer (BD Biosciences) to remove the clusters of cells.

Brachial DRGs were dissected (from E11 embryos) or whole embryonic trunks collected (E10.5 and younger) in Leibovit’z L-15 medium (Life Technologies) on ice. The tissue was incubated in 0.05% trypsin-EDTA (1×) (Life Technologies) for 5, 7, 10 and 15 min for tissue obtained from E9.5, E10.5, E11.5 and E12.5, respectively, at 37 °C, in a thermomixer comfort (Eppendorf) at 700 RPM. After spinning down the samples at 100 RCF for 5 min, the supernatant was removed and replaced by Leibovit’z L-15 medium (Life Technologies). The tissue following enzymatic digestion was physically triturated using two different sizes of pipettes previously coated with 0.2% bovine serum albumin until the solution homogenized. The cell suspension was then filtered through a 70-μm cell strainer (BD Biosciences) to remove the clusters of cells.

After establishing and expanding adherent cultures, cells were trypsinized (0.25% Trypsin-EDTA (1x), phenol red; Life Technologies) and were seeded (2×10⁴ cells/well) in 6-well ultra-low attachment (ULA) plates (Corning Inc, Corning, NY) in StemPro mesenchymal stem cell serum free medium (Life Technologies). The cells were incubated at 37°C in a humidified atmosphere at 5% CO₂ for 4–5 days to form spheroids.

The SELENOT-derived peptide 43–52 (corresponding to the sequence H-Phe-Gln-Ile-Cys-Val-Ser-Sec-Gly-Tyr-Arg-OH) designated as PSELT and its inactive form (Ser 46,49, designated as the inert-PSELT or I-PSELT) were chemically synthesized as described previously [17 (link)]. Dulbecco’s Modified Eagle Medium F-12 (DMEM/F-12), Dulbecco’s phosphate-buffered saline (DPBS), penicillin/streptomycin, 0.25% Trypsin-EDTA (1×), and fetal bovine serum (FBS), 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sodium palmitate (PA), 3-(4,5-Dimethylthiazol-)2,5-diphenyl Tetrazolium Bromide (MTT), β-nicotinamide adenine dinucleotide (NADH), and tween-20 were purchased from Sigma Aldrich (Saint Louis, MO, USA). Sulfo-Nsuccinimidyl oleate (SSO) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), and nonfat dried milk were purchased from PanReac AppliChem (Glenview, IL, USA). Before each experiment, all solutions were freshly prepared. Absolute ethanol, hydrochloric acid, and methanol were purchased from Carlo Erba Reagents (Cornaredo, Milan, Italy).

Human malignant melanoma cells (MM127,31 (link)32 (link)33 ) were cultured with 10% fetal calf serum (FCS), RPMI–1640, 2 mM L-Glutamine, 23 mM HEPES (Invitrogen, Australia) and 1% v/v penicillin/streptomycin (Invitrogen, Australia). Prior to confluence, cells were lifted using 0.05% trypsin–EDTA(1×) (Invitrogen, Australia) and viable cells were counted using a Trypan blue exclusion test and a haemocytometer.