Guave easycyte plus system

The expression of specific cell surface protein markers (SSEA-3, TRA-1–60, A2B5) on TERA2.cl.SP12 was analyzed using flow cytometry as described previously.⁵³ Briefly, TERA2.cl.SP12 cells, 2 × 10⁵ per 25 cm² flask, were seeded 12–24 h before treatment with 10 μM retinoid for 7 days. The primary antibodies used were SSEA-3 (diluted 1:10, University of Iowa Hybridoma Bank), TRA-1–60 (diluted 1:50, Abcam) and neural cell marker A2B5 (diluted 1:40, R&D Systems). Labeled cells were analyzed in a Guave EasyCyte Plus System (Millipore) flow cytometer and thresholds determining the numbers of positively expressing cells were set against the negative control antibody, P3X.

Flow cytometry was carried out on live TERA2.cl.SP12 cells, incubated at a density of 0.2 × 10⁶ cells per 25-cm² flask for 12–24 h before treatment with 10 μM retinoid for 7 days. Specific cell-surface primary antibodies were used: SSEA-3 (1:10), (University of Iowa Hybridoma Bank), TRA-1-60 (1:50), (Abcam) and neural cell marker A2B5 (1:40), (R&D Systems). Cell suspensions were centrifuged at 1000 rpm and resuspended in wash buffer (0.1% BSA in PBS) and added to 96-well plate for incubation with the primary antibodies, followed by several washes and then incubation with FITC-conjugated secondary antibody IgM (1:128) (Sigma-Aldrich). Labelled cells were analysed in a GuaveEasyCytePlus System (Millipore) flow cytometer and thresholds determining the numbers of positively expressing cells were set against the negative control antibody, P3X, a generous gift from Prof. P. Andrews, Sheffield University.