Mtt assay

Human PDLSCs were gift from Dr. Songtao Shi, and the PDLSCs were characterized as reported previously.¹⁵ Cells (passage 3–5) were thawed and seeded into a 75 cm² culture flask and cultured with a complete medium containing minimum essential medium α (α-MEM) with 10% fetal bovine serum and 1% penicillin-streptomycin. To test the effect of PPi on PDLSC proliferation, the PDLSCs were seeded at a density of 1000 cells/well in a 96-well plate. Three groups were divided according to the PPi (p8010, Sigma-Aldrich) concentrations (0, 10, and 100 μM). The cell proliferation was determined by an MTT assay (30006, Biotium) according to the manufacturer's instruction. In brief, cells were incubated with 100 μL α-MEM containing 10 μL MTT solution for 4 h at 37°C and then dissolved by 200 μL dimethyl sulfoxide (DMSO) (D2650, Sigma-Aldrich). The absorbance was measured at a wavelength of 570 nm with a microplate reader (SpectraMax 250) at the baseline of 630 nm.

To evaluate the effect of different Hex carrier proteins and endocytosis inhibitors on viability of HeLa cells, we used an MTT assay (Biotium) for measuring cell metabolic activity as an indicator of cell viability. After incubating cells with protein carriers for 24 hours or endocytosis inhibitors for 1 hour, MTT assay was conducted following manufacturer’s instructions. Absorbance at 570 nm and 630 nm of cell samples was measured by a Synergy 2 plate reader (Biotek). Background absorbance of 630 nm was subtracted from each well and all samples were normalized to the PBS-treated cells to calculate % metabolic activity. To determine the difference in fluorescence of muGFP-Hex and sfGFP-Hex, samples were diluted to various concentrations and fluorescence was analyzed with an excitation wavelength of 488 nm and an emission wavelength of 525 nm, using a Synergy 2 plate reader (Biotek).

Poietic human mesenchymal stem cells (hMSCs; Lonza, PT-2501) were grown in supplemented media (Lonza, PT-3001) and seeded in a T75 flask at a concentration of 5000 cells/cm². These cells were labeled with nanoparticles and incubated under standard conditions. The hMSCs were washed three times with PBS to remove free nanoparticles and detached using TrypLE Express (Life Technologies). Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for the MTT assay (Biotium). Then, 10 μL of MTT solution was added to 100 μL of medium in each well, mixed briefly, and incubated at 37 °C for 4 h. Then, 200 μL of DMSO was added to each well and pipetted to dissolve the resulting formazan salt. Absorbance was measured to 570 nm. The MTT assays were conducted by plating 8000 cells per well in replicate (n = 8) in a 96 well plate and treated at varying timepoints (0–24 h) at a constant concentration (0.42 mg/mL) as well as at varying concentrations (0–0.84 mg/mL) at a constant time (4 h) unless otherwise noted. The wells were analyzed in replicate (n = 8).