Celltag 700

Live cells (1 × 10⁴) were seeded in 96-well plates and incubated with various concentrations (ranging from 0.01 to 10 μM) of IRD 800-labeled aptamers at the 5′ end. This incubation took place in growth medium containing 5% FBS at 37 °C for 1 h. All oligos were purchased from Integrated DNA Technologies, Inc. (Coralville, IA).
Following the incubation period, the medium was aspirated, and the cells were washed. Fluorescence intensities were then quantified in the 800 nm channel of the LI-COR Odyssey Infrared Imaging System. Subsequently, the cells were fixed and permeabilized using a Triton washing solution, and CellTag 700 (LI-COR) stain was added. The cells were further incubated for 1 h to correct for variations in cell number from well to well. After washing, the CellTag 700 stain was detected in the 700 nm channel of the LI-COR Odyssey Infrared Imaging System. SigmaPlot software (version 14.5) was used for data plotting and curve fitting to estimate the dissociation constants (Kd), using the equation Y = BmaxX / (Kd + X), where Bmax represents maximum fluorescence intensity, Y represents fluorescence intensity at each concentration, and X represents aptamer concentration (Lin et al.,2020).

After data acquisition on the Flexstation-3 plate reader, the cells in the black 96-well plate were fixed in 4% paraformaldehyde for 1 h at RT. This was followed by permeabilization with 0.1% Triton-X (Sigma Aldrich) for 10 min at RT. The cells were washed with 1× PBS followed by incubation with CellTag 700 (LI-COR Biosciences, Lincoln, NE, USA) (1:3000) for 1 h at RT. After washing twice with 1× PBS, the plate was dried and imaged on the LI-COR imaging station (LI-COR Biosciences).

MCSs were fixed in a 10% formalin sucrose solution for 15 min at 37 °C, permeabilised in permeabilisation buffer pH 7.2 (30 mM sucrose, 50 mM NaCl, 7 mM MgCL2 (hexahydrate), 20 mM HEPES, 0.5% Triton X-100 in 1 × PBS), and blocked in 1% (w/v) milk/PBS for 1.5 hr at 37 °C on a plate shaker. MSCs were stained using primary antibodies 1:200 anti-PRUNX2 (rabbit, pS465 Abgent), ALP (sc-166261, Santa Cruz Biotechnology), OCN (sc-73464, Santa Cruz Biotechnology), OPN (sc-21742, Santa Cruz Biotechnology,) OSX (sc-22533, Santa Cruz Biotechnology), GapDH (sc-32233, Santa Cruz Biotechnology) and CD90 (13-0909-82, eBiosciences). The primary antibody incubation was carried out at 37 °C for 2.5 hr, following 5 washes in PBS/0.1% Tween. GapDH and CellTag 700 stain (Li-COR, cat: 926-41090, Lincoln, USA) were used as the reference controls. CellTag or rabbit anti-GapDH primary antibody was diluted at 1:2000. The Li-COR secondary antibodies (anti-rabbit, cat: 926-32211; anti-mouse, cat: 926-32210) were used at a dilution of 1:2000. For GapDH, the secondary antibody was diluted at a dilution of 1:5000 with a 680 nm infrared dye. The secondary antibodies were incubated at room temperature on a shaker for 1.5 hr, followed by 5 × 5 min washes in PBS/0.2% Tween. The quantitative spectroscopic analysis was carried out using the LI-COR Odyssey Sa (LI-COR, Nebraska, USA).