D2534

The effect of Docosahexaenoic acid (Sigma-Aldrich D2534) treatment was tested on hiPSC-derived MN cultured in 24-well plates differentiated from the ALS cell lines. Treatment was carried out form DIV 21 until DIV 42, by replacing half of the medium every second day, using a final concentration of 100 µM. The effect of DHA treatment was determined by measuring the levels of the phenotypic rescue using immunofluorescence.
The Tetanus neurotoxin (Sigma-Aldrich T3194) was used at a final concentration of 15 nM. The treatment was performed in hiPSC-derived MN differentiated from the Healthy I cell line, starting from DIV 42 and carried out for 24 h.

BV2 microglia were a kind gift from Dr. Jonathan Godbout and HippoE-14 neurons were purchased from Cedarlane Labs (CLU198, under MTAIN-051533). Both cell types were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin and maintained at 37°C and 5% CO₂ atmosphere unless otherwise noted. In all experiments, a 1,000x concentration of sodium palmitate (P9767, Sigma) was dissolved in molecular biology grade water heated to 70°C as previously described (Ye et al., 2016 (link); Tse and Belsham, 2018 (link)). For all experiments, a final concentration of 100 μM PA was used, which is consistent with previous studies that treated microglia, astrocytes, or neurons with palmitate (Listenberger et al., 2001 (link); Wang et al., 2012 (link); Duffy et al., 2015 (link); Frago et al., 2017 (link); Hidalgo-Lanussa et al., 2017 (link); Sergi et al., 2018 (link); Tse and Belsham, 2018 (link); Yanguas-Casás et al., 2018 (link)) and is consistent with a physiological range of plasma palmitic acid levels (Abdelmagid et al., 2015 (link)). DHA (D2534, Sigma) was dissolved in 100% molecular biology grade ethanol at 1,000x concentration and diluted to working concentration of 30 μM, which is consistent with previous concentrations used with BV2 microglia (De Smedt-Peyrusse et al., 2008 (link)).

All cell culture-related media were purchased from GE Healthcare Life Sciences (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Corning^® (Corning, NY, USA). A penicillin–streptomycin solution was purchased from TOKU-E (Bellingham, WA, USA). The n-3 PUFAs we used were α-linolenic acid (ALA, cis,cis,cis-9,12,15-octadecatrienoic acid, L2376, Sigma-Aldrich, St. Louis, MO, USA), EPA (cis-5,8,11,14,17-eicosapentaenoic acid, E2011, Sigma-Aldrich), and DHA (cis-4,7,10,13,16,19-docosahexaenoic acid, D2534, Sigma-Aldrich), and the n-6 PUFAs we used were linoleic acid (LA, 9-cis,12-cis-linoleic acid, L1376, Sigma-Aldrich) and arachidonic acid (AA, cis,cis,cis,cis-5,8,11,14-eicosatetraenoic acid, A3611, Sigma-Aldrich). Polyethyleneimine (PEI) and polybrene were purchased from Millipore (Darmstadt, Germany). A protease inhibitor cocktail was purchased from Roche (Basel, Switzerland).

PUFAs used in our experiment were prepared by conjugating either docosahexaenoic acid (Sigma-Aldrich, D2534) or arachidonic acid (Sigma-Aldrich, A3611) with fatty acid free bovine serum albumin (BSA) at 4:1 ratio and stored at −20 °C. The fatty acids were mixed with 1 mL methanol and evaporated under nitrogen gas stream. BSA/PBS solution was added to the mix, sonicated for 1 hour, and pH was adjusted to 7.4 with 1 N sodium hydroxide (NaOH).

A single administration of the either vehicle (0.2% ethanol in saline) or DHA (Sigma D2534, 250 nmoL/kg) in a volume of 5 mL/kg was carried out intravenously 30 min after the trauma to the brain. The DHA dose chosen was based on a previous study showing DHA induced functional improvements in a rat spinal cord injury model [75 (link)].