I9516

The polymeric components of the
bioreactor, as well as the PDMS channel mold were fabricated from
a heat-resistant acrylate-based photopolymer (RGD525, Stratasys) using
the Objet Eden 260 V polyjet 3D printer (Stratasys). During the manufacturing
process, overhangs and internal structures were stabilized by a support
material (SUP705, Stratasys) which was removed after printing by mechanical
ablation and dissolution in 1 M sodium hydroxide solution under constant
stirring (370 rpm, 30 °C, 12 h). The components were rinsed in
DI water and dried in ambient air. The three-way tubing connectors
were 3D-printed using a Form 3 stereo-lithography printer (Formlabs)
in combination with the corresponding high-temperature photopolymer
(FLHTAM02, Formlabs). After fabrication, the tubing connectors were
immersed in isopropanol (99.5%, Sigma-Aldrich, I9516) and were cleaned
in an ultrasonic bath for 5 min. The reactor base plate was manufactured
from a 2 mm thick aluminum plate using a milling machine (Trochoidal
Performance Cutting, VollHartMetall).

RNA was isolated from the abdominal fat bodies using Trizol LS (ThermoFisher, #10296028). 300 μL of Trizol LS was added to fat bodies in 100 μL of PBS. The tissue was homogenized with a motorized pestle for 2 minutes before adding another 600 μL of Trizol LS (3:1 mixture of Trizol LS:PBS). The resulting solution was centrifuged at 12,000g for 10 minutes at 4°C. The aqueous supernatant layer was collected in a new tube, while carefully avoiding disturbing the other layers of the phase separated solution. RNA was extracted from the aqueous supernatant layer by vigorously shaking with 240 μL of chloroform (Fisher Scientific, #C298), again carefully avoiding other layers following phase separation. The aqueous phase was transferred to a new tube and the RNA was precipitated by incubating at room temperature with 500μL of 100% isopropanol (Sigma-Aldrich, #I9516). Following centrifugation at 12,000g for 10 minutes at 4°C, the supernatant was removed leaving only the RNA pellet. The pellet was washed with 1 mL of 75% ethanol (Sigma-Aldrich, #E7023), then air dried for 5–10 minutes before resuspension in RNase–free water.

Trizol samples containing 20 µL of sorted microglia were thawed on ice before adding 200 μL chloroform (BP1145-1, Fisher Scientific). Samples were incubated at room temperature for 5 min and spun down at 14,000 rpm at 4 °C for 15 min. The top fraction was collected and incubated with an equal volume of isopropanol (I9516, Sigma) for 10 min at room temperature. The samples were spun down at 12,000 rpm 4 °C for 10 min, followed by 2 washes with 70% ethanol. After the last wash, ethanol was aspirated off, and the pellet was left to air dry for 15 min before adding 100 μL DNAse/RNAse free water. Sample quality was assessed using the NanoDrop 2000 Spectrophotometer (Thermo Scientific). cDNA synthesis was achieved using Sensifast cDNA Synthesis kits (BIO-65054, Bioline). Expression levels of Klf4 (Mm00516104_m1) were determined using the Sensifast Probe No-ROX kit (BIO-86005, Bioline) and the CFX384 real-time PCR machine (1855484, BioRad) following the manufacturing protocols.

Snap frozen liver tissue (~ 100 mg) was thawed and homogenized in 1 ml water. Next, 125ul of 50% Trichloroacetic acid (TCA, T6399, Sigma Aldrich) was added to the homogenate and incubated on ice for 20 min. After incubation, samples were centrifuged at 1000 RPM for 5 min at 4 °C. The pellets were hydrolyzed in 12 N HCl overnight at 110 °C. The dried pellets were reconstituted in water and incubated for 6 hrs at room temperature, and incubated in chloramine T solution (1.4% chloramine T, 402869, Sigma Aldrich in 0.5 M Sodium Acetate, S2889, Sigma Aldrich and 10% isopropanol, I9516, Sigma Aldrich) for 20 min at room temperature. Finally, 500ul of Ehrlichs solution (1 M p-Dimethylaminobenzaldehyde, 156477, Sigma Aldrich in 70% isopropanol and 30% perchloric acid, 311421, Sigma Aldrich) was added for 40 min at 65 °C. The absorbance of each sample was measured at 550 nm.

After MACS isolation, positive fractions were immediately lysed with 0.8 ml cold TRIzol LS (Invitrogen 10296028, ThermoFisher Scientific) and homogenized by thorough pipette and vortex mixing. After a 5 min initial incubation, 0.2 ml chloroform (C7559, Sigma) was added and samples were incubated for 2–3 min before being centrifuged for 15 min at 12,000 × g and 4°C. The aqueous phase was transferred to a new tube with 0.5 ml isopropanol (I9516, Sigma) and RNA was precipitated by incubating for 10 min at RT. After being centrifuged for 10 min at 12,000 × g and 4°C, the supernatant was discarded, and the RNA washed twice by resuspending the pellet in 75% ethanol (E7023, Sigma) in UltraPure Nuclease-free ddH₂O (Invitrogen 10977023, ThermoFisher Scientific), then vortexing briefly and then spinning down for 5 min at 7,500 × g and 4°C. After the second wash was removed, RNA was air-dried for 10 min before resuspension in 25 μl RNase-free water. Samples were then incubated at 60°C for 10–15 min before RNA quantity and purity were determined by standard spectrophotometry methods (NanoDrop 1000, ThermoFisher Scientific). If necessary, RNA samples were stored at –80°C before cDNA synthesis and qPCR analysis downstream.