Ckx41 microscope

Chondrocytes were seeded in 6-well culture plates (TCP), some of which were coated with hBMSC-ECM, at a density of 2,000 cells/cm², and cultured with fresh chondrocyte growth medium for up to 10 days. Medium was changed every 2–3 days. Cell morphology at different time points on different substrates was captured using an Olympus CKX 41 microscope equipped with a Leica camera, using LAS V4.9 software. The groups were denoted as ECM and TCP, respectively. Cell proliferation rate of chondrocytes growing on different substrates was analyzed with MTS assay (Promega, Madison, WI, USA) over a period of 10 days, as cells growing on ECM reach confluence after 7 days and those on TCP after 10 days. Briefly, at day 1, 2, 3, 4, 5, 6, 7 for ECM group and day 1, 3, 5, 7, 9, 10 for TCP group, 3 ml of diluted MTS solution was added to each of the three wells of cultures of ECM and TCP groups. After incubation in 37°C for 2h, a 200 µl aliquot of the MTS solution was aspirated from each well and transferred to a 96-well plate for measurement of absorbance at 492 using a plate reader (Biotek Synergy HT, Winooski, VT, USA). Corresponding cell numbers were calculated based on a standard curve calibrated using a set of cultures of known cell numbers.

Lizards tail samples were collected after 0, 3, 7, 14, or 21 days following original tail amputation. Samples were fixed overnight in 4% paraformaldehyde, decalcified for 1 week in 10% EDTA (pH 7.4), equilibrated to 30% sucrose, embedded in optimal cutting temperature compound (OCT, Tissue-Tek), sectioned at 7 or 20 μm thickness, mounted on glass slides, and stained with 4′,6-diamidino-2-phenylindole (DAPI). For labeling of proliferating cell populations with 5-ethynyl-2’-deoxyuridine (EdU) (ThermoFisher Scientific), animals received IP injections of EdU (50 mg/kg) four hours prior to sample collection. Samples were cryosectioned and stained according to the manufacturer’s instructions. Images were captured with an Olympus CKX41 microscope outfitted with a Leica DFC 3200 camera. To measure phagocyte levels in vivo, histology samples were collected from lizards treated with DiI liposomes and imaged with a Keyence BZ-X800 microscope as 16 μm-thick Z-stacks, and the areas of DiI signal were quantified.