Novostar fluorescence plate reader

TAMRA-Tat, TAMRA-Tat-NR2B9c, and TAMRA-Tat-N-dimer were dissolved in 0.9% NaCl and administered as a 100 µL bolus injection (3 nmol/g body weight) into the tail vein of 12-week-old male NMRI mice (27–33 g). Following 1 h of circulation, the mice were deeply anesthetized by i.p. injection of 0.05 mL/10 g body weight Ketaminol/Dexdormitor (2.25 mg ketamine/0.03 mg meteoidin per 10 g body weight in 0.9% NaCl). The chest cage was opened and the mice transcardially perfused with 0.01 M PBS (pH 7.4) prior to isolation of the brain, heart, lungs, spleen, kidney, liver, and 2 cm of the upper part of the intestine. All tissues were immediately put on ice and kept at −80 °C until analysis.
The tissues were homogenized in 1 mL of ice-cold lysis buffer using a 7-mL Dounce tissue grinder set (Merck, St. Louis, MO, USA). Homogenates were left on ice for 10 min prior to centrifugation at 14,000× g and 4 °C for 10 min. Supernatants (100 µL) were transferred to a black clear-bottom 96-well plate for quantification of the TAMRA-peptides using a NovoStar fluorescence plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission set to 542 and 568 nm, respectively. The molar peptide amounts were calculated using standard curves that were freshly prepared in lysates from relevant blank tissue at concentrations ranging from 30 nM to 2 µM.

Absorbance and fluorescence spectra of MFF in 10 mM Tris–HCl buffer containing 0.1% SDS, pH 8.0 were obtained at 23 °C using a Shimadzu UV-1800 UV-VIS Spectrophotometer and a Photon Technology International Quanta Master spectrofluorometer (HORIBA Scientific), respectively. Quenching of MFF (4 μM) by FAS and FeCl₃ was measured using a NOVOstar fluorescence plate reader (BMG Labtech). Aqueous FAS was prepared with equimolar ascorbic acid to prevent oxidation. Tris buffer was treated with Chelex (1421253, BioRad) overnight to remove trace heavy metals. Fluorescence spectra were uncorrected for lamp intensity and detector sensitivity.