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The cells were washed three times with secretion buffer (4.5 mM KCl, 138 mM NaCl, 4.2 mM NaHCO 3 , 1.2 mM NaH 2 PO 4 , 2.6 mM CaCl 2 , 1.2 mM MgCl 2 , and 10 mM HEPES, which was adjusted to pH 7.4 with NaOH) supplemented with 0.1% fatty acid-free bovine albumin serum (BSA

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This is why aliquoting, minimizing air exposure, and following recommended storage temperatures are critical after reconstitution

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