NPs were conjugated with equal mole of Alex Flour 750 (AF750) through reaction with AF750 NHS Ester (ThermoFisher Scientific) in PBS (pH 7.4). Unreacted dye was removed through dialysis. NPs were injected via the tail vein 10 mins after CCI surgery (n=5 mice per group). Each group blood (20 μL) was collected at 5, 15, 30 mins and 1, 3, 6, 12, 24, 36, 48 hrs after injection using a heparinized pipette. Blood was stored in an EP tube with 20 μL heparin (50 U/mL), mixed, and centrifuged at 3,000 g for 10 mins at 4°C. The plasma was collected after centrifuge. Plasma AF750 concentration was quantified based on AF750 fluorescence and plotted with time. Fluorescence intensity was detected immediately with a microplate reader (Synergy™ Mx, BioTek Instruments, Inc.).
Af750 nhs ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AF750 NHS Ester is a fluorescent labeling agent used for the covalent attachment of dyes to primary amines found in proteins and other biomolecules. It provides a bright, photo-stable fluorescent signal that can be detected using near-infrared (NIR) imaging systems.
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Lab products found in correlation
2 protocols using af750 nhs ester
1
Pharmacokinetics of NP-bound AF750
2
Synthesis and Purification of FH-AF750
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FH-AF750 was synthesized using previous published methods [17 (link)]. Briefly, washed and purified Feraheme was incubated with hydroxybenzotriazole (HOBT) and N-(3-dimethyaminopropyl)-N′-ethylcarbodiimide (EDC) in MES buffer (pH 6.0–6.4) at room temperature for 20 min. Ethylenediamine (1 M) was added to the mixture and incubated at 50 °C for 90 min. This raw FH–amine solution was then purified on a Sephadex G-25 column, and concentrated using 50 kDa Amicon centrifugation filters. This purified FH–amine compound then underwent conjugation with an AF750-NHS ester (Thermo Fisher Scientific, Waltham, MA, USA). The final product, FH-AF750, was again purified in a Sephadex G-25 column and reconcentrated by 50 kDa Amicon centrifugation filters. Nanoparticle (NP) concentration, and the payloads of AF750/NP of the final product, were determined spectrophotometrically.
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