A6003

Female Cnp-Mek1DD, Cnp-DR, or Plp-DR and control littermate mice were
euthanized by CO₂ inhalation at 8-13 weeks of age. Spleens
were removed and immediately placed in 2 mL DMEM on ice, and
dissociated as described previously with modifications (Pachynski et al.,
2015). Spleens were chopped and 1 mL of digestion medium
in HBSS added (0.04 mg/mL collagenase I, LS004214, Worthington
Biochemical; 1.67 mg/mL collagenase IV, LS004188, Worthington
Biochemical; 30 mg/mL DNAse I, D4527-40KU, Sigma) for 25 min.
incubation at 37°C, then 5 mL of DMEM were added and suspension
filtered through a 70 µm filter. Cells were centrifuged at 450 g for
6 min., then erythrocytes were lysed using 8.29 g/L NH₄Cl,
1 g/L KHCO₃, and 37.2 mg/L EDTA for 4 min. After adding
12 mL of DMEM, suspensions were filtered through a 70 µm filter,
washed with 10 mL DMEM, and centrifuged at 450 g for 6 min. Cells were
resuspended in 20 mL of HBSS, centrifuged at 450 g for 5 min., then
resuspended in 1 mL HBSS for cell counting with trypan blue. After
cell counting with a 1:4 dilution, cells were centrifuged at 450 g for
5 min. and resuspended at 1 x 10⁶ cells/50 uL of FACS
buffer (2% goat serum, G9023, Sigma; 1% BSA, A6003, Sigma; in HBSS).
Only female mice were used for all immune cell experiments in order to
match EAE experiments.

Isolated crypts (4000, estimated 1 million cells) were plated on a 24 well plate coated with 10% Matrigel (Corning 356231 growth factor reduced) in 400 μL crypt medium (RPMI (GIBCO, no glucose, 11879020) basal medium with 5mM glucose) and allowed to incubate in a humidified 37°C incubator at 5% CO₂ for 15 min. 10% essentially fatty acid free BSA (Sigma Aldrich, A6003) in PBS was complexed at a volume ratio of 6.7:3 with palmitic acid-[9,10-³H] (Pekin Elmer, NET043001MC) by vortexing for 60 s and was added at a 1:100 ratio to crypt medium. This hot medium was split into half and Etomoxir (75 μM final) was added to inhibit FAO. 100 μl of the hot FFA:PA:medium mixture was added to the incubated crypts, for a total volume of 500 μl, and were incubated for 1 hour, 37°C, 5% CO₂. Samples were removed from the wells and pelleted at 21K rcf. for 2:30 min. 400 μL of resulting supernatant was transferred to a filter column (Fisher Scientific, 11-387-50) containing 3 mL of activated Dowex®1X8 resin (Sigma Aldrich, 217425). 2.5 mL of ddH₂O was added to elute ³H-water from the column. 750 μL of eluent was added to 2.5 mL of EcoLume (MP Biomedicals, 882470). Betacounts were measured on a scintillation counter (Beckman Coulter, LS6500).

The apex of the heart was homogenized using a glass dounce homogenizer at 4°C for 3 minutes in isolation buffer (200 mM Mannitol [M9647; Sigma], 60 mM sucrose [S7903; Sigma], 5 mM KH₂PO₄ [P5379; Sigma], 5 mM MOPS [M1254; Sigma], 1 mM EGTA [E4378; Sigma], and 3 mg of Nagarse protease [P8038; Sigma]). After 3 minutes, isolation buffer with 0.1% w/w BSA (A6003; Sigma) was added and the homogenate was centrifuged at 8000 × g for 10 min at 4°C. The pellet was resuspended in isolation buffer with BSA and centrifuged at 8000 × g for 10 min at 4°C. The resulting pellet was resuspended in isolation buffer with BSA and centrifuged at 700 × g for 10 min at 4°C. The supernatant was collected and centrifuged at 8000 × g for 10 min. The pellet was resuspended in isolation buffer with BSA and mitochondrial concentration was measured using a citrate synthase (CS) assay as previously published by Oroboros.³¹ Briefly, purified mitochondria were placed in a solution containing 100 mM Tris pH 8.1 (T1503, Sigma), 0.25% triton X-100 (T8532, Sigma), 0.31 mM acetyl CoA (A2181; Sigma), 0.1 mM DTNB (D218200, Sigma), and 0.5 mM oxaloacetate (04126, Sigma) at 30°C. Immediately, absorbance was measured at a wavelength of 412 nm.