Bullet blender gold

Internal standard (20 μL of 1 μg·mL⁻¹ solution of 25CN-NBOMe-d₃) was added to each media sample (200 μL) and ammonium bicarbonate solution was used to adjust the sample’s pH to 8.4 approximately. The resulting aqueous phase was extracted with 400 μL of ether twice, and combined organic extracts were evaporated to dryness. The dry residue was reconstituted in 200 μL of 5% (v/v) methanol in 0.1% formic acid and this solution was transferred into an HPLC vial.
C. elegans mycelium was thawed and cut into small pieces. Approximately 300 mg of wet material, 100 mg of bullets for homogenization (Next Advance ZrOB05—yttria-stabilized zirconium oxide beads, 0.5 mm dia.), and 1 mL of 0.1% formic acid were added to the weighted Eppendorf tube. Each sample was thoroughly vortexed and homogenized using the Bullet Blender Gold (Next Advance) at 4 °C (10 min, speed 8). Consequently, the samples were centrifuged for 10 min at 13,200 RPM at 4 °C (Eppendorf Centrifuge 5415 R), the resulting supernatants were separated and their pH was adjusted to 8.4 approximately by addition of ammonium bicarbonate solution. The aqueous phase was extracted with 400 μL of ether three times and combined organic extracts were evaporated to dryness. The dry residue was reconstituted in 300 μL of 5% (v/v) methanol in 0.1% formic acid and this solution was transferred to an HPLC vial.

DMP cross-linked beads were gently well-mixed in 1 mL of bead wash buffer then pipetted in 100 μL aliquots (0.5 mg beads/sample) into Sarstedt 2.0 mL LB microtubes. Monkey heart tissue samples (20 mg to 200 mg) were weighed and transferred to a Sarstedt 2.0 mL LB microtube. The tube was washed and dried, accurately re-weighed, and the amount of heart tissue recorded. RIPA lysis buffer (500 μL) containing the protease inhibitor cocktail was added to heart tissue followed by approximately, 30–50 stainless steel beads (0.9–2.0 mm). Homogenization was conducted using the Bullet Blender Gold homogenizer (Next Advance, Troy, NY) at a speed of 10 for 5 min at 4 °C. Samples were then lysed completely by additional probe sonication on ice with 30 pulses at power 4 using a sonic dismembranator (Fisher Scientific, Pittsburgh, PA). The SILAC-hFXN-M internal standard solution (20 μL of 2 μg/mL, 40 ng) was then added to each sample and appropriate amounts of hFXN-M standards 4 ng to 200 ng (4, 10, 15, 20, 30, 40, 80, 100, 150, 200 ng) in 5% BSA solution for preparation of the standard curve^{13 (link)}. Supernatants were removed from the DMP cross-linked beads and tissue and BSA standard samples added into the Sarstedt 2.0 mL LB microtubes containing the DMP cross-linked beads. Samples were then incubated with the beads at 4 °C overnight on the rotator.

Lipid standards were purchased from Avanti Polar Lipids (Avanti Polar Lipids Inc., AL, USA). Solvents for MS analysis and for tissue extraction were LC-MS grade and obtained from Sigma-Aldrich Co. 20 mg of tumor and non-neoplastic stroma tissues were homogenized in water using a bullet blender homogenizer (Bullet Blender Gold, Next Advance, Inc., Averill Park, NY, USA) in the presence of zirconium oxide beads (0.5 mm) at speed 8 for 5 min at 4° C. A portion of the homogenate (corresponding to 2 mg wet weight) was immediately subjected to a one-phase methanolic lipid extraction [40 (link)]. The homogenate was sonicated in 1 ml MeOH containing 0.001% butylated hydroxytoluene (as antioxidant) in a bath sonicator for 5 min, then shaken for 5 min and centrifuged at 10,000 g for 5 min. The supernatant was transferred into a new Eppendorf tube. The extracts were stored at –20° C. MS analysis was performed by an LTQ-Orbitrap Elite instrument (Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nano ion source TriVersa NanoMate (Advion BioSciences, Ithaca, NY, USA). Lipid classes and species were annotated using the lipid classification system [41 (link)] and individual lipids were identified by LipidXplorer software [42 (link)].

Total RNA was isolated from GD/ED 10 mouse conceptuses using the RNeasy Plus Mini Kit (Qiagen; Germantown, MD) following homogenization in lysis buffer using the TissueMiser (Fisher Scientific; Waltham, MA) or Bullet Blender Gold (Next Advance; Troy, NY). A minimum of 3 conceptuses were pooled per dam. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, CA). Relative transcript abundance was measured using Power SYBR® Green Supermix (Applied Biosystems; Foster City, CA) with the C1000 Touch Thermal Cycler (CFX96 Real Time Systems; Bio-Rad, Hercules, CA). Each sample was assayed in duplicate for all target and housekeeping genes. Average threshold cycle (Ct) values were normalized to average Ubc^{78 (link),79 (link)} Ct values. Relative transcript abundance of each gene of interest was determined using the ΔΔCt method^{80 (link)}. Briefly, transcript expression in individual mice is presented relative to the mean expression value in Mal− mice. When possible, primer pairs were designed to span large introns such that amplification exclusively represented cDNA template. Primer sequences and amplicon sizes are listed in Supplemental Table 1.

To separate the synaptic and extra-synaptic regions, diaphragms were incubated in ice-cold diethyl pyrocarbonate (DEPC)-treated PBS with BTX for 30 min, and dissected based on NMJ staining under a conventional IF microscope. Muscles were submerged in Trizol (Ambion, Carlsbad, California) and homogenized using Bullet Blender Gold (Nextadvance BB24-AU, Averill Park, New York). RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and prepared for RT-qPCR analysis or RNA-sequencing experiments. First-strand complementary DNA was synthesized from 200 ng of RNA using the SuperScript First-Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, California). RT-qPCR was performed on a Step One Plus Real Time PCR machine (Applied Biosystems, Foster City, California), with Platinum SYBR Green qPCR SuperMix-UDG and ROX master mix (Invitrogen) using primers against AChE, Chrna1, Chrnd, Chrne, Etv5, Musk and B2M (Supplementary file 1). All reactions for RT-qPCR were performed using the following thermal cycler conditions: 50°C for 2 min, 95°C for 2 min, 40 cycles of a two-step reaction, denaturation at 95°C for 15 s, annealing at 60°C for 30 s. Variant expression was normalized to B2M using the comparative C_t method and reported relative to the average of the synaptic samples.