Figure 5.

scASCs display reduced NOX1 expression, reduced ROS accumulation and long-term expansion. aASCs (Abd) and scASCs (SC) were cultured under normoxic conditions (21% oxygen) and the following analyses were performed at the indicated passages (P). (a) ROS accumulation was detected by DCFDA staining and analyzed by FACS (fluorescence-activated cell sorting) (aI) and by fluorescence microscopy (aII). (aIII) The mRNA expression level of different antioxidant enzymes and various cytokines was evaluated by quantitative reverse transcription-PCR (qRT-PCR) at the indicated passages. Error bars represent S.D. (bI) Cells at passages 3–5 were seeded at a density of 2 × 10⁴ cells per 6 cm plate. The division rate was assessed by calculating the logarithmic growing phase. (bII) Growth curves of ASCs from an abdominal fat source (dotted lines) and from subcutaneous fat (continuous line). Only scASCs cells were able to undergo long-term expansion. Experiments were performed on five independent preparations from each fat source. Error bars represent S.D. **P<0.01 and *P<0.05 (Student's two-tailed t-test for equal variance). (cI) A percentage of apoptotic cells from aASCs and scASCs was evaluated by PI/Annexin staining and analyzed by FACS. (cII) The results represent the fold change of Annexin-positive cells from aASCs (black bars) and scASCs (gray bars). Experiments were carried out in two independent ASC preparations. Error bars represent S.E. **P<0.01 and *P<0.05 (Student's two-tailed t-test for unequal variance). (cIII and cIV) The percent of dead cells was evaluated by aqua blue staining at the indicated passages. (cV) Activities of caspase-3/7 in aASCs and scASCs were evaluated by a Caspase-Glo 3/7 Assay Kit. (d) RNA expression level of NOX1 and various cytokines was evaluated by qRT-PCR at the indicated passages. Error bars represent S.D. of subcutaneous ASCs (S.C) and abdominal ASCs (Abd). Untreated aASCs were cultured in parallel to both scASCs and to aASCs that were treated with an NOX1 inhibitor and are therefore used as control in both Figures 4 and 5