TY - JOUR
T1 - Neutral red assay modification to prevent cytotoxicity and improve reproducibility using E-63 rat skeletal muscle cells
AU - Hall, Jeffery O.
AU - Novakofski, Jan E.
AU - Beasley, Val R.
PY - 1998/7
Y1 - 1998/7
N2 - 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 μg/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 μg/ml. Primary quail myoblasts developed lysosomal swelling and ruptured more rapidly than E-63 cells when exposed to NR at either 127 or 157 μg/ml. For confluent 10-day cultures of E-63 cells exposed to NR at 127 μg/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.
AB - 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 μg/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 μg/ml. Primary quail myoblasts developed lysosomal swelling and ruptured more rapidly than E-63 cells when exposed to NR at either 127 or 157 μg/ml. For confluent 10-day cultures of E-63 cells exposed to NR at 127 μg/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.
KW - Colorimetric assay
KW - Cytotoxicity
KW - E-63 muscle cells
KW - Neutral red
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U2 - 10.3109/10520299809141112
DO - 10.3109/10520299809141112
M3 - Article
C2 - 9735880
AN - SCOPUS:0031721885
SN - 1052-0295
VL - 73
SP - 211
EP - 221
JO - Biotechnic and Histochemistry
JF - Biotechnic and Histochemistry
IS - 4
ER -