Anti α6 integrin apc

LEPC cluster derived and enriched mesenchymal cells (LMSC, P1) were isolated from 3 organ-cultured corneoscleral buttons (mean age, 66.5 ± 4.6 years) and characterized by flow cytometry using anti-CD90, -CD73, -CD105, -CD44, -CD34, -CD45, -CD31 and isotype control antibodies (BD Biosciences, Heidelberg, Germany). LEPC were characterized using anti-α3 integrin-APC, anti-α6 integrin-APC, anti-β1 integrin-FITC and anti-β4 integrin-eFluor660 antibodies (eBioscience, Frankfurt, Germany). Single cell suspensions (0.5–1 × 10⁶ cells) were incubated with saturating concentrations of respective primary antibodies or conjugated antibodies in 100 µl PBS, 0.1% sodium azide and 2% fetal calf serum for 40 min. After three washes, the cells were centrifuged at 200 × g for 5 min. Primary antibody reactions were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG for 30 min. Cells were then washed and re-suspended in ice-cold PBS. After addition of 5 µl of 7-amino-actinomycin D (7-AAD) to exclude dead cells, cytometry was performed on a FACSCanto II (BD Biosciences) by using FACS Diva Software. A total of 10,000 events were acquired to determine the positivity of cell surface markers.

Primary isolated OC were cultured in vitro and passaged for about 6-8 times. The OCs were harvested and then incubated with anti-EpCAM-APC, anti-Sca-1-PE, anti-CD44-PE, anti-CD49f-APC, anti-CD45-APC, anti-CD34-PE, anti-c-kit-FITC, anti-α6 integrin-APC, anti-β1 integrin-PercpCy5.5, anti-β4 integrin-PercpCy5.5 (all from ebioscience, San Diego, CA) at 4°C for 30 mins, after that, cells were fixed with 1% paraformaldehyde and analyzed by a BD Calibur (Becton, Dickinson and Company, New Jersey) and Flowjo software (Tree Star, San Carlos, CA).