Xi71 inverted microscope

Spinning disk confocal microscopy was performed using an Improvision UltraVIEW VoX system (Perkin-Elmer) built around a Olympus XI71 inverted microscope, equipped with PlanApo objectives (×60, 1.45 NA) and controlled by the Volocity software (Improvision). To image DiI and SiR, 561-nm and 640-nm laser lines with appropriate filters (615 ± 35 and 705 ± 45 nm, respectively) were used.

Persisters were enriched as described before and resuspended in a final volume of 20 µl MgSO₄. After flushing the channels of the microfluidic device with MgSO₄, cells were loaded by syringe injection followed by chip centrifugation. A peristaltic pump was used to flow medium through the device at a flow rate of 90 µl/min. The microscope chamber, which also contained the medium reservoir, was constantly held at 37 °C. Images were obtained using an Olympus XI71 inverted microscope with a 100x objective.
Live/dead staining was performed by flowing propidium iodide into the channels of the microfluidic device for 10 min at a concentration of 30 µM.

The late osteogenic marker was calcium nodules. It was examined according to the instructions of Alizarin Red Staining solution (Beyotime, Shanghai, China). The cells were cultured as described in 2.5.1 for 7, 14, and 21 days. They were fixed with 95% ethanol for 10 min, and the washed by PBS for three times. The staining procedure was terminated with distilled water after adding appropriate Alizarin Red Staining solution for 30 min. Calcium nodules were observed with a XI71 Inverted Microscope (Olympus, Japan). A semi-quantitative analysis of the expression levels of calcium nodules was also carried out. Briefly, 500 μL of 10% Hexadecane-pyridinium Chloride solution (Solarbio, Beijing, China) was added to the plates containing calcium nodules. The liquid was added to 96 wells at 100 ul per well after 15 min (n = 3). The OD value was read at 506 nm, and the semi-quantitative calcium nodules in each group were evaluated.