A microassay for measuring glycogen in 96-well-cultured cells.
This study describes a rapid, sensitive, and automated spectrophotometric enzymatic microassay that measures the intracellular glycogen of primary cultured hepatocytes and other cultured cells in 96-well plates and can be adapted for other samples that are transferred to these plates. The procedure involves in situ disruption of cells, followed by hydrolysis of glycogen into glucosyl units by fungal glucoamylase (exo-1.4-alpha-glucosidase, EC 3.2.1.3), and glucose determination with the glucose oxidase colorimetric method. The color intensity can be measured in conventional ELISA readers, and the data can be fed to an on-line computer for rapid processing. The advantages of this method are its simplicity and automation, the reduction in sample handling, and the small number of cells required compared to other conventional methods.
Gómez-Lechón
M J
MJ
Unidad de Hepatología Experimental, Hospital Universitario La Fe, Valencia, Spain.
Ponsoda
X
X
Castell
J V
JV
D000818:Animals; D002236:Carbohydrate Conformation; D002478:Cells, Cultured; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose / Q000032:analysis*; D006868:Hydrolysis; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008112:Liver Glycogen / Q000032:analysis*; D008297:Male; D008832:Microchemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D008112:Liver Glycogen; D004247:DNA; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose