5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium bcip nbt

The cells were collected using a lysis buffer (0.5 M Tris-hydrogen chloride (HCL), pH 8.8, containing 0.9% sodium chloride, 1% Triton X-100, and 200 mM ethylenediaminetetraacetic acid (EDTA)), and ALP activity was measured using 1-Step™ p-nitrophenyl phosphate (Sigma) according to the manufacturer's recommendations. ALP-positive cells were stained with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT; Sigma) at room temperature. TRAP activity and staining were processed using an Acid Phosphatase Kit (Sigma) in accordance with the manufacturer's instructions.

To examine the effect of BMP-2 on osteogenic differentiation of MSCs, 9.5 × 10³ MSCs at passage 4 were cultured in 24-well plate (Corning, USA) with osteogenic differentiation medium supplemented with 100 ng/ml of BMP-2 (R&D systems, USA) for 3, 7, 14, 21, and 28 days. The osteogenic differentiation was measured by alkaline phosphatase staining using 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT; Sigma-Aldrich, USA). Briefly, the cultured cells were washed with PBS and fixed with 4% paraformaldehyde for 5 min at 4°C. Then, BCIP®/NBT® liquid substrate (Sigma-Aldrich, USA) was added and incubated for 30 min at a room temperature. The cells were wash twice with distilled water and observed under inverted microscope (Nikon TS100, Japan). MSCs cultured with complete medium and osteogenic differentiation medium were used as controls.

Antibody-secreting cells were measured in peripheral blood according to our previously described enzyme-linked immunosorbent spot (ELISPOT) procedure (26 (link), 37 (link)). Briefly, nitrocellulose (bottom) plates (MSHAN-4550; Millipore, Bedford, MA) were coated with 100 μl of sialidase (25 μg/ml) in sodium bicarbonate buffer (pH 9.6), affinity-purified goat anti-human immunoglobulin (5 μg/ml; Jackson Immuno Research, West Grove, PA) to detect the total number of circulating ASCs, or keyhole limpet hemocyanin (KLH; 2.5 μg/ml [Pierce Biotechnology, Rockford, IL]) in PBS as a negative control. After blocking with RPMI solution, isolated PBMCs were incubated on the plates for 3 h, and secreted antibodies were detected with peroxidase-conjugated mouse anti-human IgA (1:500 dilution; Southern Biotech, Birmingham, AL) and alkaline phosphatase-conjugated IgG (1:500 dilution; Southern Biotech, Birmingham, AL). ASCs were detected with 3-amino-9-ethylcarbazole (AEC) (AEC premix solution; Sigma-Aldrich) and 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (BCIP/NBT; Sigma-Aldrich). The antigen-specific IgA and IgG isotypes of ASCs were expressed as the frequencies of the total circulating ASCs of the same isotype at the same time point.

Cells were washed with PBS and lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 50 mM NaF, 5 mM EDTA, 0.5% w/v sodium deoxycholate, and 1% Triton X-100) containing phosphatase and protease inhibitors. The protein concentrations of the lysates were determined using the BCA method. Aliquots of the lysates containing 50 μg of protein were mixed with SDS-PAGE sample buffer and subjected to SDS-PAGE followed by western blotting using rabbit polyclonal antibody directed against human carboxylesterase 1 and alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, CA, USA). Immunoreactivity was detected using 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium BCIP/NBT (Sigma, St Louis, MO, USA).

Cells and spheroids were fixed with 4% Paraformaldehyde (Sigma) at room temperature for 30 min. This was followed by washing with 1X PBS and Tris buffered saline (100 mM Tris and 5 mM MgCl2 in deionized water, pH 7.4). Further, the cells and colonies were incubated with the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (Sigma Aldrich) at room temperature for 2 h. The appearance of pinkish brown colonies was monitored over time to avoid oversaturation. The reaction was terminated by removing the substrate solution followed by PBS wash. The bright field images were then acquired using an EVOS FL Cell Imaging System (Thermo Fisher Scientific). To segment spheroids in a bright field image, local contrast was enhanced, followed by a variance and Gaussian filtering and then thresholding (Fig. S1B). In order to obtain the alkaline phosphatase activity/intensity, color deconvolution was done using the vectors (r = 0.65, g = 0.4, b = 0.64). A segmented spheroid with mean alkaline phosphatase activity > 0.5 was considered to be positive (Fig. S1F). These steps were carried out in Fiji^{33 (link)}.