We fasted 6- to 8-week-old mice for 4 h and then each mouse received 2 µg per g body weight BODIPY and 2 µg per g body weight rhodamine-PEG (Methoxyl PEG Rhodamine B, MW 5,000 g mol−1) with 0.2% fatty acid–free BSA by gavage. We collected feces from 20 min to 4 h after BODIPY was administered. We homogenized 50 mg of feces in PBS containing 30 mM HEPES, 57.51 mM MgCl2 and 0.57 mg ml−1 BSA with 0.5% SDS and sonicated for 30 s; we then centrifuged at 1,000g for 10 min. We transferred supernatants to 96-well plates and measured fluorescence values immediately using a fluorescence microplate reader for endpoint reading (Molecular Devices). We also measured serum BODIPY and rhodamine concentrations from blood drawn 4 h after gavage. For both measurements, we subtracted baseline fluorescence from untreated mice from measured fluorescence. For BODIPY, the excitation and emission wavelengths were 488 nm and 515 nm, respectively. For rhodamine-PEG, the excitation and emission wavelengths were 575 nm and 595 nm, respectively.
Fluorescence microplate reader for endpoint reading
Manufactured by Molecular Devices
The Fluorescence microplate reader for endpoint reading is a laboratory instrument designed to measure the fluorescence intensity of samples in a microplate format. It is capable of performing endpoint fluorescence measurements, providing quantitative data on the fluorescent properties of the samples.
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2 protocols using fluorescence microplate reader for endpoint reading
1
In Vivo Absorption Kinetics of BODIPY and Rhodamine
2
Oral Gavage Uptake of 2NBDG and Rhodamine-PEG
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We fasted 6- to 8-week-old mice for 4 hr and then each mouse received an oral gavage of 200 μl olive oil or 2 μg per g body weight 2NBDG and 2 μg per g body weight rhodamine-PEG (Methoxyl PEG Rhodamine B, MW 5,000 g mol−1) with 0.2% fatty acid–free BSA by gavage. We collected feces from 20 min to 4 hr after 2NBDG was administered. We homogenized 50 mg of feces in PBS containing 30 mM HEPES, 57.51 mM MgCl2 and 0.57 mg ml−1 BSA with 0.5% SDS and sonicated for 30 s; we then centrifuged at 1000 g for 10 min. We transferred supernatants to 96-well plates and measured fluorescence values immediately using a fluorescence microplate reader for endpoint reading (Molecular Devices). We then subtracted baseline fluorescence from untreated mice from measured fluorescence. We also measured enterocytes’ 2NBDG content after isolation of primary cells as described above, using excitation and emission wavelengths of 488 nm and 515 nm, respectively. For rhodamine-PEG, the excitation and emission wavelengths were 575 nm and 595 nm, respectively.
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