Dulbecco s phosphate

PD sheets and TKA sheets were washed in Dulbecco’s phosphate-buffered saline (DPBS; Gibco). The sheets were then incubated in TripLE Express® (Gibco) at 37 °C for 15 min and centrifuged at 1500 rpm for 5 min. The cell sheets were resuspended in 0.25 mg/mL Collagenase P (Roche, Basel, Switzerland) at 37 °C for up to 30 min and then centrifuged at 1500 rpm for 5 min. The isolated cells were finally resuspended in DMEM/F12, and cell count and viability were determined using the trypan blue exclusion assay.

The effect of maternal age on the expression of multipotency and pluripotency markers in PDMSCs was analyzed by flow cytometry. PDMSCs were washed with Dulbecco’s phosphate-buffered saline (DPBS, Gibco) and resuspended in fluorescence activated cell sorting (FACS) buffer (Thermo Fisher Scientific) with FITC-conjugated anti-human CD90, PE-conjugated anti-human CD73, and APC-conjugated anti-human CD105 (BioLegend, CA, USA) for 20 minutes at room temperature in the dark. The cells were then washed with FACS buffer and resuspended in fixation buffer (Thermo Fisher Scientific). For intracellular staining, the cell membrane was permeabilized with 1x Perm buffer (BioLegend) for 20 minutes at room temperature. The cells were resuspended in 1x Perm buffer with PE-conjugated anti-human OCT4, FITC-conjugated anti-human SSEA4 and Alexa Fluor® 647-conjugated anti-NANOG antibodies for 2 hours in the dark. The cells were then washed and resuspended in fixation buffer. The fluorescence intensity of 20,000 cells was recorded on a flow cytometer (Guava, Merck) and analyzed using FlowJo software with excitation/emission wavelengths 494/520 for FITC, 496/578 for PE, 650/660 for APC and 650/660 for Alexa Fluor® 647.

Bacteria were grown in BaLi medium and washed twice with Dulbecco's phosphate-buffered saline (DPBS; Gibco). Bacterial binding to purified plasma, cellular, and Rn Fn was performed using 5 × 10⁸ bacteria (optical density [OD]; 1.0 OD = 5 × 108 cells/mL) and 7.5 μg Fn in DPBS. For cross-linking analysis, samples were incubated for 30 min at 37°C while gently shaking. For competition binding experiments, 0.5 or 5 μg/μL heparin were added and incubated at 37°C for 2 h while shaking. After each incubation step, cells were centrifuged and washed three times with DPBS.

PD sheets were washed in Dulbecco’s phosphate-buffered saline (DPBS; Gibco). The sheets were then incubated in TripLE Express® (Gibco) at 37 °C for 15 min and centrifuged at 1500 rpm for 5 min. The cell sheets were resuspended in 0.25 mg/mL collagenase P (Roche, Basel, Switzerland) at 37 °C for up to 30 min and then centrifuged at 1500 rpm for 5 min. The isolated cells were finally resuspended in DMEM/F12, and the cell count and viability were determined using the trypan blue exclusion assay.