4 nitrophenyl phosphate disodium salt

Alkaline phosphatase activity (ALP) was measured in lysates from cells that were cultured with mineralization medium. Cells were harvested at days 0 and 14 of culturing. Cells were washed with PBS and lysed with 200 μL Milli-Q water and were frozen in −20°C for storage. After three freeze-thaw cycles, samples were collected by scraping. ALP was measured according to the method described by Lowry (45 (link)), using 4-nitrophenyl phosphate disodium salt (Merck, Darmstadt, Germany) at pH 10.3, as a substrate for ALP. Absorbance was measured at 405 nm with a Synergy HT spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). DNA was measured in the same lysate using CyQuant Cell Proliferation Assay Kit (Molecular Probes, Leiden, The Netherlands). Fluorescence was measured at 485 nm excitation and 528 nm emission with a Synergy HT spectrophotometric microplate reader. Alkaline phosphatase was expressed as μmol/ng DNA.

Cells were harvested at day 0, 3, 7, 14, and 21 of culturing. In order not to sample dead cells, the adherent cells were washed twice with PBS. Next, they were lysed in 150 μL MilliQ per well and stored in -20°C. Prior to analysis, the plates underwent three cycles of freeze-thawing. ALP activity was measured using 4-nitrophenyl phosphate disodium salt (Merck, Darmstadt, Germany) at pH 10.3 as a substrate for ALP according to the method described by Bastidas-Coral (Bastidas-Coral et al., 2016 (link)). After incubation of 60 min at 37°C, the reaction was stopped with sodium hydroxide. Absorbance was measured with 405 nm with a Synergy HT spectrophotometer (BioTek Instruments Inc., Winooski, VT, United States). DNA concentration (ng/mL) was measured using CyQuant Cell Proliferation Assay Kit (Molecular Probes, Leiden, Netherlands) mixed with lysis buffer. Fluorescence was measured at 485 nm excitation and 528 nm emission with a Synergy HT spectrophotometer (BioTek Instruments Inc., Winooski, VT, United States). ALP activity was expressed as ALP per DNA (nMol/ng).

hASC and hOBs cultured for 1 or 7 days with PMMA were lysed with 500 μL lysis buffer and stored at −20°C prior to use. ALP activity was measured in the cell lysate using 4‐nitrophenyl phosphate disodium salt (Merck, Darmstadt, Germany) at pH 10.3 as a substrate, according to the manufacturer's instructions. The absorbance was read at 405 nm with a Synergy HT® spectrophotometer. ALP activity was quantified against a standard curve of 4‐nitrophenol solution and expressed as μmol 4‐nitrophenol per ng DNA.

hASCs cultured for 48 h, 4 days, and 7 days with proinflammatory and anti-inflammatory cytokines were lysed with CyQuant lysis buffer. ALP activity was measured in the cell lysate using 4-nitrophenyl phosphate disodium salt (Merck, Darmstadt, Germany) at pH 10.3 as a substrate for ALP, according to the method described by Lowry [27 (link)]. The absorbance was read at 405 nm with a Synergy HT spectrophotometer. ALP activity was expressed as μM per ng DNA.

Purified pili (5 μg/mL, sample A) were dissolved in 50 mM sodium carbonate buffer (pH 9.6) and coated overnight onto ELISA plates (X50 Immunolon 4HBX plates, Thermo Scientific) at 4°C. For the lectin probing using the mannose-specific lectin HHA, mannan was used as a positive control (5 μg/mL). In the case of the fucose-specific lectin AAL, Lewis X was coated as a positive control (5 μg/mL) (Lectinity). Wells only containing 50 mM sodium carbonate buffer were used as negative controls for the assay. The next day the wells were washed trice with PBS, preceding the blockage of the wells with 0.5% PVA dissolved in TBS [43 (link)] for 2-3h at 37°C. The wells were then washed with PBS prior to the addition of the digoxigenin-labeled lectins dissolved in TBS + 0.5% PVA (1h, 37°C). Unbound lectins were removed by washing the wells with PBST (PBS + 0.1% Tween 20). Then the secondary antibody, anti-digoxigenin (Roche) [44 (link)] was added to the cells for 1 hour at 37°C. Prior to development, the wells were washed with PBST. 4-nitrophenyl phosphate disodium salt (1mg/mL, Sigma-Aldrich^®) dissolved in substrate buffer (carbonate bicarbonate buffer, pH 9.6) was added to each well. Color was allowed to develop for 15–30 min at room temperature and the absorbance was measured at 405 nm.

ALP is an enzyme expressed by cells during osteogenesis and is well established as a differentiation marker. A colorimetric assay was used to measure the ALP expression. This reaction used 8 mg/mL of 4-nitrophenyl phosphate disodium salt (Sigma-Aldrich) dissolved in tris-hydrochloric acid solution.
This essay consisted of filtering the medium that was in contact with the samples and reading the absorbance at 405 nm to obtain the baseline. Then, the ALP solution was added in a 1:1 ratio to the medium. This solution was incubated for 30 min, and the absorbance was measured.
The statistical analysis performed to check the significance between each sample and their growth during the experience was the same as the one described for the adhesion and proliferation assay.