Formamide

After the second IMS wash, 250 μL of elution buffer comprised of 5 vol % DNase/RNase free UltraPure™ water (Invitrogen by Life Science, Waltham, MA, USA), 45 vol % formamide (Thermo Scientific, Waltham, MA, USA) and 50 vol % 80 mM NaOAc (sodium acetate) was used to remove the polyG oligos from the bead assay. Sodium acetate was used as an electron mediator during the oxidation of polyG. Samples containing the elution buffer and beads were heated to 90 °C for 10 min, and then transferred to a three-electrode 96 well plate (Cat. No. DRP-96x550, DropSens, Asturias, Spain) placed in a Faraday cage. The working, counter, and reference electrodes were comprised of carbon, carbon and silver, respectively. Square wave voltammetry (SWV) was used to detect the redox current density of the polyG oligo tag. An EmStat3+ potentiometer, along with PalmSens (PsTrace v5.3) software, was used to measure the current density of the redox reaction using square wave voltammetry (SWV). SWV measurements were taken between 0.4–1.2 V at a scan rate of 500 mVs⁻¹.

A 0.5% sterile Evans blue (Sigma) solution was prepared in PBS and passed through a 0.2 μm filter to remove any particulate matter that has not dissolved. Mice having undergone either sham or CCI surgery were injected with 200 μL Evans blue either intravenously (i.v.) or i.p. No differences were observed between using i.v. or i.p. administration of Evans blue (not shown). Anesthetized sham or CCI injured animals received intracardiac perfusion with PBS, pH 7.4, 3 h after the Evans blue injection to remove any excess dye. Brains were removed and the ipsilateral and contralateral hemispheres were incubated separately in 500 μL Formamide (Thermo Fischer) for 24 h at 55 °C. Samples were centrifuged to pellet the tissue and the supernatant absorbance was measured at 610 nm using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). Formamide was used to blank the instrument. Evans blue absorbance was normalized to the contralateral hemisphere for each animal.

We purchased 21, 18, 13, and sex chromosome aneuploidy detection kits (fluorescent PCR capillary electrophoresis) from Sun Yat‐sen University Daan Gene Co., Ltd. and Guangzhou Darui Biotechnology Co., Ltd.; formamide, Liz600, ABI3500DX sequencing instrument, and corresponding Gene‐Mapper 5.0 software from Thermo Fisher Scientific Co., Ltd.; and the K5800 microspectrophotometer from Beijing Keao Company.

Evans Blue dye was dissolved in normal saline at 0.32 mg/mL, followed by filtration through a 0.2 μm filter. Mice were injected i.p. with dye-containing saline and euthanized at 1 hour afterward. Peritoneal fat tissues were collected, weighed, and then incubated in formamide (Thermo Fisher Scientific) at room temperature for 48 hours to extract Evans Blue dye. Extracted dye was measured by reading absorbance at 620 nm using a Spark microplate reader (TECAN). Extracted dye was quantified by using a standard curve of Evans Blue dye concentration, and calculated as amount of dye (ng) per mg of dry tissue weight.

To quantify lung permeability, fluorescein isothiocyanate (FITC)-dextran, 4 kDa (Sigma-Aldrich) was intravenously injected via the retroorbital sinus at 20 mg/kg in a total volume of 100 μL.
Fluorescence was read in BALF and serum samples using the Synergy™ Mx microplate reader (Biotek Instruments) at 492 nm (emission) and 515 nm (detection). The amount of FITC-dextran retained in the lung was also measured. To that end, the right lungs were immersed in Formamide (ThermoFisher Scientific, Cornellà de Llobregat, Spain) at 37 °C. After 24 h, the samples were centrifuged at 15,000× g for 10 min, and the supernatants’ fluorescence was measured at the aforementioned wavelengths. Fluorescence values were normalized to the weight of each lung at the moment of the sacrifice.