Janus green whole cell stain

Analogously to the single-target labeling procedure described above, multiplexed quantitative analysis on the same sample was achieved by first incubating cells with a cocktail of three pre-assembled 1′Ab/PrA-ssDNA probes (i.e., anti-Lamin A Ab/PrA-X1, anti-HSP90 Ab/PrA-Y1, and anti-Cox4 Ab/PrA-Z1) for 1 h, followed by labeling with a cocktail of three pre-assembled ssDNA′-AP probes for 1 h and thorough washing with TBS. Individual ssDNA′-AP probes were then released into solution via sequence-specific 10-min displacement with complementary probes X3, Y3, and Z3 and processed for measurement with a plate reader. To validate the accuracy of relative protein levels obtained from the same sample through the DNA displacement method, each of the model targets (Lamin A, HSP90, and Cox4) was individually labeled with respective 1′Ab/PrA-ssDNA and ssDNA′-AP probes in separate cell samples and processed for measurement with a plate reader without displacement, as in a typical in-cell ELISA. To account for differences in cell counts between specimens in this case, measurements were normalized by total cell numbers using Janus Green whole cell stain (Thermo Scientific).

The FGF23 levels in the cultured cell supernatants were evaluated by ELISA Kit (KAINOS laboratories, Japan). The minimal detectable concentration is 3 pg/mL. The FGF23 levels of MSCs supernatant were compared to OS (control positive), PODO (control negative), and cells culture media. Replicate background measurements were subtracted to all 450 nm measures. To normalize, Janus Green Whole-Cell Stain (Thermofisher) was added for 5 min. Careful washing was followed by addition of elution buffer and absorbance was read at 630 nm. The resulting A450–A630 values were then normalized to the A615 values to account for differences in numbers of cells.