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The fluorescence intensity representing the cholesterol was examined using a microplate reader at an excitation wavelength of 482 nm and an emission wavelength of 515 nm

Cell pellets (~2030 l of packed cell pellet) were re-suspended in 1 ml buffer containing 20 mM HEPES (pH = 7.5), 10 mM KCl, 250 mM sucrose, 5 mM MgCl 2 , 1 mM EGTA, 1x phosphatase inhibitor cocktail (Sigma, P0044) and 1x protease inhibitor cocktail (Sigma, P8340), and lysed by addition of digitonin (Sigma, D141), 200 g/ml final concentration at 4 C for 10 min

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HGH effects accumulate, HbA1c moves slowly, and fluid symptoms can show up only after exposure is already excessive