Hall J O, Novakofski J E, Beasley V R
Department of Animal, Dairy and Veterinary Sciences, College of Agriculture, Utah State University, Logan 84322-5700, USA.
Biotech Histochem. 1998 Jul;73(4):211-21. doi: 10.3109/10520299809141112.
Cellular uptake of neutral red dye (NR) is currently used as an indirect measure of viable cells in cultures. We used E-63 rat skeletal muscle cells to identify causes of NR assay variability and to develop modifications that substantially reduce it. Three methods of NR preparation and/or addition to cells were used. When NR medium was prepared, incubated overnight, and filtered to remove precipitates, the amount of dye precipitated varied greatly. Coefficients of variation (CVs) in NR uptake were greater than 25% between assays. Higher NR concentrations, longer incubation times, increased pH, and decreased temperature promoted NR precipitation in media. NR media prepared and filtered just prior to use or direct addition of prefiltered NR stock solution to cell cultures resulted in much smaller CVs between assays. NR was cytotoxic to E-63 rat muscle and primary quail myoblasts in a time- and concentration-dependent manner. NR exposure to E-63 cells for greater than 1.25 and 2 hr at 157 or 127 microg/ml, respectively, was associated with swelling and rupture of lysosomes. By contrast, there was no evidence of cytotoxicity when E-63 cells were exposed to NR for 1 hr at either 127 or 157 microg/ml. Primary quail myoblasts developed lysosomal swelling and ruptured more rapidly than E-63 cells when exposed to NR at either 127 or 157 microg/ml. For confluent 10-day cultures of E-63 cells exposed to NR at 127 microg/ml for 1 hr, the CVs within assay and between assays were 3.3-3.9% and 5.1%, respectively. For similarly exposed, actively replicating 3-day cultures of E-63 cells, the CVs within and between assays were 6.2-9.6% and 2.4%, respectively. NR uptake by the E-63 cells was linear with respect to viable cell number.
中性红染料（NR）的细胞摄取目前被用作培养物中活细胞的间接测量方法。我们使用E - 63大鼠骨骼肌细胞来确定NR测定变异性的原因，并开发出能大幅降低变异性的改进方法。使用了三种NR制备和/或添加到细胞中的方法。当制备NR培养基、过夜孵育并过滤以去除沉淀时，沉淀的染料量变化很大。不同测定之间NR摄取的变异系数（CVs）大于25%。较高的NR浓度、较长的孵育时间、升高的pH值和降低的温度会促进培养基中NR沉淀。在使用前刚制备并过滤的NR培养基，或直接向细胞培养物中添加预过滤的NR储备溶液，会使不同测定之间的CVs小得多。NR对E - 63大鼠肌肉和原代鹌鹑成肌细胞具有时间和浓度依赖性的细胞毒性。分别以157或127微克/毫升的浓度让NR暴露于E - 63细胞超过1.25小时和2小时，与溶酶体肿胀和破裂有关。相比之下，当E - 63细胞以127或157微克/毫升的浓度暴露于NR 1小时时，没有细胞毒性的证据。当以127或157微克/毫升的浓度暴露于NR时，原代鹌鹑成肌细胞比E - 63细胞更快地出现溶酶体肿胀和破裂。对于在127微克/毫升的浓度下暴露于NR 1小时的汇合10天的E - 63细胞培养物，测定内和不同测定之间的CVs分别为3.3 - 3.9%和5.1%。对于类似暴露的、活跃增殖的3天E - 63细胞培养物，测定内和不同测定之间的CVs分别为6.2 - 9.6%和2.4%。E - 63细胞对NR的摄取与活细胞数量呈线性关系。
