Osteopontin

hMSCs were cultured on test graphene substrates for 24 and 72 hours in the absence or presence of electrical stimulation and then detached with Trypsin (Invitrogen, Carlsbad, CA). After adding complete media to deactivate the Trypsin, cells were collected by centrifugation. Cells were then washed with FACS buffer (PBS without Ca/Mg²⁺, 1% FBS, 0.1% NaN₃) twice and fixed in 1% paraformaldehyde at room temperature for 10 minutes. The cells were then permeabilized using FACS buffer with 0.1% saponin; washed once in FACS buffer; and then centrifuged to a pellet. The supernatant was discarded. Class III β3 tubulin (MAB1637, EMD Millipore, Billerica, MA) and Osteopontin (AB8448, Abcam, Cambridge, MA) were diluted 1:100 in FACS buffer plus 0.1% Triton-X 100 and treated on the cells (50μl /sample). The cells were incubated with primary antibodies for 1 hour at 4°C; washed once in 1 ml FACS buffer plus 0.1% Triton-X 100; and centrifuged; and the supernatant was discarded. Secondary antibodies specific to the primary IgG isotype were diluted in FACS buffer plus 0.1% Triton-X 100 with a final sample volume of 100μl at 1:1000 dilution. The cells were incubated for 60 minutes in the dark at 4°C; washed in FACS buffer plus 0.1% Triton-X 100; and resuspended in 300μl FACS buffer for analysis. Results were analyzed using FlowJo v8.5.2 (FlowJo LLC, Ashland, OR).