5 bromo 2 deoxyuridine

All experiments were conducted in FVB mice, both male and female and repeated at least twice and in most cases 3–4 times. For all experiments, date of birth was considered ‘Day 0’ unless otherwise specified. At day 10, mice were given four intraperitoneal BrdU (5-Bromo-2′-deoxyuridine, Roche, Mannheim, Germany) injections at a dose of 3 mg per 100 g of body weight, or EdU (5-ethynyl-2′-deoxyuridine, Invitrogen, Carlsbad, CA) injections at a dose of 10 mg/100 g of body weight 12 hours apart. The chase period for the label retaining assay was 8 weeks, and at this time point the mice were anesthetized via an intraperitoneal injection with Avertin (240 mg/kg, Sigma, St Louis, MO) and euthanized by exsanguination for collection of the salivary glands. All mice were maintained and treated in accordance with protocols approved by the University of Arizona Institutional Animal Care and Use Committee (IACUC). A total of 5 animals (3 males and 2 females) were labeled with BrdU and 46 animals (24 males and 22 females) were labeled with EdU. Mice were distributed to different experiments as indicated in the following sections.

Optic nerves were evaluated in vivo by BrdU incorporation (5-bromo-2′-deoxyuridine; Roche), as previously described (17 (link)). To evaluate OPC differentiation into mature CC1⁺ oligodendrocytes, we injected BrdU (i.p., 100 mg/g body weight) in P20 mice for 3 consecutive days and analyzed mice at P30. Briefly, cryosections were fixed in cold methanol, treated with 2N HCl for 15 minutes at 37°C, and neutralized with 0.1 M sodium borate (pH 8.5) for 10 minutes. Slides were then incubated overnight with anti-NG2 polyclonal antibody or anti-CC1 mAb and anti-BrdU mAb to identify proliferating cells at P20 and differentiated oligodendrocytes at P30. After staining with secondary antibodies, nuclei were counterstained with DAPI (Vectashield, Vector Laboratories). Quantification was performed by counting of the absolute number of positive cells per area (mm²) in 5 adjacent images per nerve section, in 2–3 serial sections (at least 4000 cells per animal).

To screen the FDA-approved drug library (Enzo Life Sciences Inc., Farmingdale, NY, USA), MSCs were seeded (1.0 × 10⁴ cells per well) onto a 96-well plate and treated with 10 µM of drug library. After 72 h of incubation, the CellTiter-Glo^® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) was used, according to the manufacturer’s instructions to investigate the change in viability. For secondary screening, MSCs were seeded (1.0 × 10⁴ cells per well) onto a 96-well plate and were treated with 10 μM and 100 μM of 6 drug candidates. After 72 h of incubation, a 5-bromo-2′-deoxyuridine (BrdU) incorporation enzyme-linked immunosorbent assay (ELISA), (Roche, Basel, Switzerland) was performed according to the manufacturer’s instructions. To investigate the optimal concentration of ethionamide, MSCs were treated with varying concentrations of ethionamide, and the BrdU ELISA was performed as described above.

T cell proliferation was measured by quantification of 5-bromo-2’deoxyuridine (Roche, Basel, Switzerland) incorporated in dividing cells using an anti-BrdU antibody. In brief, CD8+ T cells were isolated from frozen PBMCs after resting overnight in IMDM supplemented with 10% human serum (HS, Sigma-Aldrich) by negative selection using the MACS CD8+ T cell isolation kit (Miltenyi, Bergisch Gladbach, Germany). Isolated CD8+ T cells were plated at a concentration of 2 × 10⁵ cells/well and stimulated with 2% Leucoagglutinin PHA-L (1 μg/mL, Sigma-Aldrich) in a final volume of 200 μl/well of flat bottom 96-well microtiter plates. BrdU was added 24 hours prior to harvest for each time point. Incorporated BrdU was quantified by ELISA in accordance to the instructions of the manufacturer (Roche) with medium alone as control.

Cell proliferation was assayed in paraffin-embedded kidney tissue by incorporation of 5-bromo-2-deoxyuridine (Roche Molecular Biochemicals, Mannheim, Germany), as previously described [29 (link)]. Pregnant mice received an intraperitoneal injection of BrdU (100 mg/g of body weight) 2 h prior to sacrifice. BrdU-positive cells were identified using an anti-BrdU peroxidase-conjugated antibody (Roche Molecular Biochemicals, Mannheim). Immunoreactivity was visualized using Aminoethyl Carbazole horseradish peroxidase chromogen/substrate solution (Vector, USA). Apoptosis was assessed in paraffin-embedded kidney tissue using the cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) and visualized using 3,3’-Diaminobenzidine (DAB) substrate solution (Vector, USA).

Cells were seeded into 96-well microplates in sextuplicate (3 × 10³ cells per well). After incubation at 37 °C for 24 h, cells were shifted to serum-free conditions and were treated with darifenacin (0.01, 0.1, 1, and 10 µM) for 48 h. Then, cell viability and proliferation were assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; Sigma-Aldrich) and BrdU (5-bromo-2′-deoxyuridine; Roche, Mannheim, Germany) assays, respectively, according to the manufacturers’ protocols.