A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation.
A mixture of crude collagenase and non-specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünsch's substrate and protein content with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggest that collagenase enzymes do not play a major role in heart cell isolation. On the other hand, an important factor in heart digestion seems to consist of some fractions of the proteases present in crude collagenase. It is also noted that crude collagenases do not present any sensitivity to added calcium but because this ion is important to obtain isolated cells its role is discussed. According to our results, the SDS-PAGE method can be used to determine the appropriate enzyme concentrations to obtain calcium-tolerant myocytes. These myocytes have electrophysiological properties as reported in the literature.
Le Guennec
J Y
JY
Laboratoire de Physiologie des Cellules Cardiaques et Vasculaires, CNRS EP 21, Faculté des Sciences, Tours, France.
Peineau
N
N
Esnard
F
F
Lacampagne
A
A
Gannier
F
F
Argibay
J
J
Gauthier
F
F
Garnier
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D002469:Cell Separation; D017364:Collagenases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D011402:Pronase / Q000032:analysis*
D017364:Collagenases; D011402:Pronase