Department of Biobehavioral Nursing and Health Systems, School of Nursing, University of Washington, Seattle, WA 98195, USA.
Wound Repair Regen. 2010 May-Jun;18(3):335-40. doi: 10.1111/j.1524-475X.2010.00587.x. Epub 2010 Apr 29.
In wound healing studies that investigate therapeutic interventions, it is important to characterize cellular responses. In a randomized trial enrolling patients at risk for surgical infection, one goal is to phenotype cells within a polytetrafluoroethylene implant using flow cytometry and immunohistochemistry, together with standard hematoxylin- and eosin-based histology. Subcutaneous implants are removed 8-9 days postoperatively. To obtain single cells associated with the mechanism of wound healing, we initially used a mouse skin digestion protocol. We optimized this to increase the cell yield and isolate sufficient cells for flow cytometry. The modifications increased the total cells recovered per subject from an average of 5.3 x 10(4)-41 x 10(4) with an average viability of 80%. The immunoflourescent staining assay was verified for our samples, which have smaller cell sample numbers than tissue biopsies. Thirty-two samples were stained. Cells from the polytetrafluoroethylene tubes were isolated and stained positively with fluorescent-labeled antibodies to CD3, CD20, CD31, CD34, CD68, CD133, and VEGF receptor type 2. Flow cytometry data correlated with immunohistochemistry data especially with respect to CD68. This antigen was the most prevalent in both the cell analysis methods. Our findings demonstrate that flow cytometry can be used with polytetrafluoroethylene samples as an additional evaluation method to document and describe cellular wound healing responses.
在研究治疗干预措施的伤口愈合研究中，描述细胞反应非常重要。在一项招募有手术感染风险的患者的随机试验中，目标之一是使用流式细胞术和免疫组织化学，结合标准的苏木精和伊红染色组织学，对聚四氟乙烯植入物中的细胞进行表型分析。术后 8-9 天取出皮下植入物。为了获得与伤口愈合机制相关的单细胞，我们最初使用了一种小鼠皮肤消化方案。我们对此进行了优化，以增加细胞产量并分离足够用于流式细胞术的细胞。这些改进使每个受试者的总细胞回收率从平均 5.3 x 10(4)-41 x 10(4)增加，平均活力为 80%。我们的样品数量比组织活检少，因此对免疫荧光染色检测进行了验证。对 32 个样本进行了染色。聚四氟乙烯管中的细胞被分离出来，并与 CD3、CD20、CD31、CD34、CD68、CD133 和血管内皮生长因子受体 2 的荧光标记抗体阳性染色。流式细胞术数据与免疫组织化学数据特别与 CD68 相关。这种抗原在这两种细胞分析方法中都最为常见。我们的研究结果表明，流式细胞术可用于聚四氟乙烯样本，作为一种额外的评估方法，用于记录和描述细胞伤口愈合反应。
