MSC were serum-starved (0.5% FBS) overnight and pretreated for 30 min with antibodies (10 μg/mL) specific to integrin α6 (sc-6597, Santa Cruz Biotechnology), integrin β1 (sc-9970, Santa Cruz Biotechnology), integrin α2β1 (sc-59955, Santa Cruz) or a non-specific isotype control antibody (ab81032, Abcam). Cells were then rinsed with PBS, trypsinized and suspended in 2% alginate gels at 5×106 cells/mL.
Hydrogel disks were formed with an 8 mm biopsy punch and placed in 24 well plates containing growth media (α-MEM containing 10% FBS and 1% P/S). Media were collected on day 7 after refreshing the media 24 hours prior to collection. Cell content within the gel was measured by quantifying DNA. VEGF concentration within conditioned media was quantified by ELISA.
Hydrogel disks were formed with an 8 mm biopsy punch and placed in 24 well plates containing growth media (α-MEM containing 10% FBS and 1% P/S). Media were collected on day 7 after refreshing the media 24 hours prior to collection. Cell content within the gel was measured by quantifying DNA. VEGF concentration within conditioned media was quantified by ELISA.