Wigg Alan J, Phillips John W, Wheatland Loretta, Berry Michael N
Department of Medical Biochemistry, Flinders University, Bedford Park, South Australia 5042, Australia.
Anal Biochem. 2003 Jun 1;317(1):19-25. doi: 10.1016/s0003-2697(03)00057-5.
The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.
传统上，对新鲜分离的肝细胞制剂的细胞浓度和活力评估是使用血细胞计数板手动计数并进行台盼蓝拒染染色来完成的。尽管这种评估方法简单快速，但人们对这些方法的准确性仍存在担忧。虽然有简单的流式细胞术技术可用于测定细胞浓度和活力，但令人惊讶的是，这些技术尚未在分离的肝细胞制剂中得到广泛应用或验证。因此，我们研究了使用TRUCOUNT管和碘化丙啶染色的流式细胞术来测量悬浮状态下分离的大鼠肝细胞的细胞浓度和活力。与手动细胞计数相比，使用TRUCOUNT管进行分析能提供更准确、可重复的细胞浓度测量结果。使用碘化丙啶评估的肝细胞活力与所有测量的肝细胞完整性和功能指标（乳酸脱氢酶泄漏、细胞色素P450含量、细胞ATP浓度、氨和乳酸清除、尿素和白蛋白合成）的相关性比台盼蓝拒染更紧密。我们得出结论，流式细胞术技术可用于测量分离的肝细胞制剂的细胞浓度和活力。这些技术简单、快速，比手动细胞计数和台盼蓝染色更准确，且结果不受含蛋白质培养基的影响。
