The cellular uptake assay was
performed 48 h post-transduction on a heated (37 °C) orbital
shaker plate. For a 3 min preincubation, transport buffer (500 μL
of HBSS with 4.17 mM NaHCO3 and 25 mM HEPES adjusted to
a pH of 7.4 with NaOH) replaced the medium in the wells. After the
buffer was removed, cellular uptake began when 125 μL of the
test substrate solution was added into the wells. The uptake was stopped
during the linear uptake phase by aspiration of the test solution
(specific test times mentioned in the results). Three times wash with
500 μL of ice-cold transport buffer followed this step, and
the cells were lysed with 125 μL of 0.1 M NaOH [2′,7′-dichlorofluorescein
(DCF) and estrone sulfate] or 150 μL of a 3:1 methanol/water
mixture (rosuvastatin samples). Fluorescence measurement (excitation
500 nm, emission 528 nm, and bandwidth 5 nm) using the multimode microplate
reader Varioskan LUX (Thermo Fisher Scientific, Vantaa, Finland) of
the cell lysates was performed to quantify the DCF. The [3H]-estrone sulfate-containing cell lysate was neutralized with equivalent
moles of 1 M HCl before adding Optiphase HiSafe 3 scintillation liquid
and measuring radioactivity of samples using a MicroBeta2 2450 Microplate
Counter (PerkinElmer, Waltham, Massachusetts, USA). 10 μL of
the cell lysate was mixed with 300 μL of the Coomassie Plus
reagent and used for total protein amount quantification with absorbance
analysis (595 nm) on Varioskan LUX. Rosuvastatin was quantified on
a Sciex 5500Qtrap LC/MSMS system (ABSciex, Framingham, MA, USA) interfaced
with an electrospray ionization (ESI) source as described previously.29 (link)
performed 48 h post-transduction on a heated (37 °C) orbital
shaker plate. For a 3 min preincubation, transport buffer (500 μL
of HBSS with 4.17 mM NaHCO3 and 25 mM HEPES adjusted to
a pH of 7.4 with NaOH) replaced the medium in the wells. After the
buffer was removed, cellular uptake began when 125 μL of the
test substrate solution was added into the wells. The uptake was stopped
during the linear uptake phase by aspiration of the test solution
(specific test times mentioned in the results). Three times wash with
500 μL of ice-cold transport buffer followed this step, and
the cells were lysed with 125 μL of 0.1 M NaOH [2′,7′-dichlorofluorescein
(DCF) and estrone sulfate] or 150 μL of a 3:1 methanol/water
mixture (rosuvastatin samples). Fluorescence measurement (excitation
500 nm, emission 528 nm, and bandwidth 5 nm) using the multimode microplate
reader Varioskan LUX (Thermo Fisher Scientific, Vantaa, Finland) of
the cell lysates was performed to quantify the DCF. The [3H]-estrone sulfate-containing cell lysate was neutralized with equivalent
moles of 1 M HCl before adding Optiphase HiSafe 3 scintillation liquid
and measuring radioactivity of samples using a MicroBeta2 2450 Microplate
Counter (PerkinElmer, Waltham, Massachusetts, USA). 10 μL of
the cell lysate was mixed with 300 μL of the Coomassie Plus
reagent and used for total protein amount quantification with absorbance
analysis (595 nm) on Varioskan LUX. Rosuvastatin was quantified on
a Sciex 5500Qtrap LC/MSMS system (ABSciex, Framingham, MA, USA) interfaced
with an electrospray ionization (ESI) source as described previously.29 (link)
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