Biostation im q microscope

hB cells (1 × 10⁶ cells) and hMSCs (0.1 × 10⁶ cells) were added in 2 mL onto 35-mm culture dishes (BD Biosciences) and cultured for 24 h. hB cell apoptosis was determined in three ways. First, hB cells were stained with anti-CD19 antibody conjugated with APC and then with FITC-annexin and propidium iodide for 15 min (FITC-Annexin V Apoptosis Detection Kit, BD Biosciences). hB cells were analyzed using a flow cytometer (FACSCalibur) and the data were processed using Cell Quest Pro software (BD Biosciences) 38 (link). Second, hB cells were stained with anti-CD19 antibody conjugated with APC and then labeled with FITC-ApoStat (Intracellular Caspase Detection ApoStat kit, Bio-Techne, Minneapolis, MN, USA) for 1 h. hB cells were analyzed using a flow cytometer (FACSCalibur) and the data were processed using Cell Quest Pro software 39 (link). Third, CellEvent Caspase-3/7 Green ReadyProbes Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) was directly added to the co-culture of hB cells and hMSCs 40 (link). Time-lapse imaging was performed with a Biostation IM-Q microscope (Nikon) and images were acquired in two channels (phase contrast and green filter) every 10 min for 24 h 4 (link). Images were analyzed by using Imaris software 9.3.0. Green-fluorescent cells were considered apoptotic.

To track cell movement, the fibroblast cells on the gauzes were initially stained with NucBlue™ Live ReadyProbes™ Reagent (Molecular Probes, Waltham, MA, USA) at 20 °C for 20 min. Then, the stain solution was removed and 2 µl of fresh medium was added to maintain the cell culture during evaluation. The cell sheet samples on the gauze were placed on tissue culture dishes with the cell sheets facing down. Afterwards, time-lapse images of the cell migration on the gauze were acquired on Biostation IM-Q microscope (Nikon Inc, Melville, NY) with a 10X magnification (numeric aperture 0.5) and taken every 10 min at various spots for 72 h at 37 °C in a 5% CO₂ humidified atmosphere. The determination of the mean nonoverlapped velocity (n = 5) and trajectory of the cells were analyzed using CL-Quant software version 3.30 (Nikon Corporation, Minato-ku, Tokyo, Japan).

In transwell assay, B cells (100 μl) were added to the upper wells of transwell plates with a 5 μm insert (Corning, Tewksbury, MA, USA). Various concentrations of chemokines or MSCs were added to the lower wells in 600 μl of complete RPMI 1640 medium. The number of B cells that had migrated to the lower well over 1.5 h was counted using a flow cytometer [14 (link)].
For time-lapse imaging, MSCs (70 μl of 0.3 × 10⁶ cells/ml) were seeded into the left chamber and B cells (70 μl of 1 × 10⁶ cells/ml) into the right chamber of culture-insert μ-Dish^35mm culture dishes (ibidi GmbH, Martinsried, Germany). Time-lapse imaging was performed with a BioStation IM-Q microscope equipped with a 10x magnification objective (numeric aperture 0.5) in an environmental chamber kept at 37°C and 5% CO₂ (Nikon Inc., Melville, NY, USA). Dishes were preincubated for 3 h in the chamber, and then, inserts were carefully removed. Images were acquired every 2 min for 12 h [18 (link)].