Department of Chemical Engineering, Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath BA2 7AY, UK.
Biotechnol Lett. 2010 Feb;32(2):215-22. doi: 10.1007/s10529-009-0136-9. Epub 2009 Oct 11.
The interactions of post-culture treatments reagents used for fixing, lysing and cell quantification on poly(lactide-co-glycolide) (PLGA) flat sheet membrane scaffolds are presented. Lysing with Alkaline buffer solution/Triton X-100/MilliQ water (ATM) and fixing with 10% Neutral Buffered Formalin (10% NBF) had no affect on membrane structure while fixing with 95% ethanol caused smoothing of the surface, shrinkage and a reduction in surface area of 55, 48 and 33, for 100:0, 75:25 and 50:50 (PLA:PGA), respectively. PicoGreen assay was selected for cell (560pZIPv.neo) quantification since the background noise would not affect readings for cell numbers over 3,000 cells/cm(2), while the background reading was too high for MTT and Methylene Blue (MB). MB at 0.5% (w/v) was, however, deemed suitable for visualising cell morphology on the membranes. Furthermore ATM buffer was suitable for the PicoGreen assay, which allows the same samples to be used for quantification of alkaline phosphatase activity.
介绍了用于固定、裂解和细胞定量的后培养处理试剂与聚（丙交酯-乙交酯）（PLGA）平片膜支架的相互作用。用碱性缓冲溶液/Triton X-100/MilliQ 水（ATM）裂解和用 10%中性缓冲福尔马林（10% NBF）固定对膜结构没有影响，而用 95%乙醇固定会使表面光滑、收缩，对于 PLA:PGA 分别为 100:0、75:25 和 50:50，表面积减少 55%、48%和 33%。由于背景噪声不会影响超过 3000 个细胞/cm(2)的细胞数量的读数，因此选择 PicoGreen 测定法用于细胞（560pZIPv.neo）定量，而对于 MTT 和亚甲蓝（MB），背景读数过高。然而，0.5%（w/v）的 MB 被认为适合观察膜上的细胞形态。此外，ATM 缓冲液适用于 PicoGreen 测定法，该测定法允许对碱性磷酸酶活性进行定量的相同样品进行使用。
