We are trying to perform whole cell patch-clamp using rat’s atrial cardiomyocytes in accordance wit the paper below: Am J Physiol Heart Circ Physiol 293: H2448–H2461, 2007; doi:10.1152/ajpheart.00032.2007.
In this paper, authors recommended to use papain for cell isolation and suggested to dissolve papain into dithiothreitol(DTT) instead of the solution recommened in the product information; a solution containing 1.1mM EDTA, 0.067mM mercaptoethanol and 5.5mM cysteine-HCl.
Does anyone know the advantage of use DTT for this purpose? Is this important for cell isolation?
Papain is soluble in water to form a stock solution. Immediately prior to use , the enzyme is typically diluted in buffer containing 5mML -cysteine. The powder as supplied should be stored at -20 degrees Celsius. Although papain solutions have good temperature solution stability is pH dependent.
The main advantage of using DDT in cell isolation is that it does not affect humans or animals (rat). DDT accumulates in the fatty tissues of animals and humans but produces no known toxic effects while it is stored in the fat. DDT is metabolized into various breakdown products in the body including DDE, DDD4 and DDA5. Hence it is suitable for cell isolation.
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