Poly(ethylene glycol)-ε-poly(caprolactone) (PEG-PCL) block copolymer with molecular weight 5–5.5 kDa was purchased from Polymer Source (Montreal, QC, Canada). PX866 and wortmannin were purchased from LC laboratories (Woburn, MA). SN-38 was purchased from Selleckchem (Houston, TX). Acetonitrile (ACN), ethanol and water were purchased from Fisher Scientific (St. Louis, MO). PEG-PCL (100 mg, dissolved in ethanol) was mixed with drugs (10 mg, dissolved in ethanol) in a round bottom flask. The mixed solution was evaporated at reduced pressure to remove solvent and create a thin film. Polymer and drug were then reconstituted in water and sonicated for 5 min to form micelles. The micelle solution was centrifuged to remove free drug and other insoluble impurities. The supernatant containing drug-loaded nanoparticles was removed and collected by freeze-drying. The diameter and surface charge of PNPs were determined by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern, UK), and polydispersity index (PDI) of particle size was calculated by the equipped software. Alexa Fluor 546™ NHS Ester (Succinimidyl Ester) was purchased from Thermo Fisher Scientific (Waltham, MA).
Alexa fluor 546 nhs ester succinimidyl ester
Manufactured by Thermo Fisher Scientific
Alexa Fluor 546™ NHS Ester (Succinimidyl Ester) is a fluorescent labeling reagent used for covalent attachment of fluorescent dyes to primary amines on proteins and other biomolecules. It is a bright, photostable dye with an excitation maximum at 556 nm and an emission maximum at 573 nm.
Automatically generated - may contain errors
4 protocols using alexa fluor 546 nhs ester succinimidyl ester
1
Preparation of PEG-PCL Nanoparticles for Drug Delivery
2
In Vivo Lymphatic Remodeling Assay
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Skin samples from C57BL/6J Prox-1GFP mice were dissected from areas of aberrant lymphatics (equivalent areas used in sham control mice). Lymphatic vessels were visualized using Prox-1GFP epifluorescence under a fluorescence stereo-dissecting microscope with an eGFP filter (Leica Microsystems). Between 15 and 30 images were taken per mouse, blinded, and lymphatic channels measured for aperture in ImageJ. All image measurements were pooled per mouse to calculate average lymphatic widths.
BmL3 were washed before incubation with 50 μM Alexa Fluor 546 NHS ester (succinimidyl ester) (Thermo Fisher Scientific) in Fluorobrite DMEM (Thermo Fisher Scientific) for 2 hours. C57BL/6J Prox-1GFP transgenic mice were injected with 400 fluorescent BmL3 as described above. After 3 hours and 1–6 dpi in mice, areas of subcutaneous tissues where lymphatic remodeling occurs were imaged as above (DsRed and eGFP filters).
BmL3 were washed before incubation with 50 μM Alexa Fluor 546 NHS ester (succinimidyl ester) (Thermo Fisher Scientific) in Fluorobrite DMEM (Thermo Fisher Scientific) for 2 hours. C57BL/6J Prox-1GFP transgenic mice were injected with 400 fluorescent BmL3 as described above. After 3 hours and 1–6 dpi in mice, areas of subcutaneous tissues where lymphatic remodeling occurs were imaged as above (DsRed and eGFP filters).
3
Anti-TNF-α and Anti-MCP-1 Antibody Protocol
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mAb1 (cAb) and mAb11 (dAb) anti-TNF-α antibodies were purchased from Biolegend. Human TNF-α protein was purchased from R&D Systems (210-TA). Human blood was purchased from BioIVT. Unlabeled mouse anti-human MCP-1 antibody (clone 5D3-F7; BDB551226), unlabeled mouse anti-human MCP-1 antibody (clone 10F7; BDB555055), and recombinant human MCP-1 (BDB554620) were purchased from BD Biosciences. The SiteClick biotin antibody labeling kit, Alexa Fluor 546 NHS ester (succinimidyl ester) and Alexa 647 NHS ester (succinimidyl; ester), and all other chemicals were purchased from Thermo Fisher Scientific. All chemicals were of analytical grade and used without further purification. 1% v/v Vectabond was purchased from Vector Laboratories. PEG succinimidyl valerate MW-5000 (mPEG-SVA) and biotin-PEG-SVA MW-5000 (Biotin-PEG-SVA) were purchased from Laysan Bio. Custom imaging chamber components were purchased from Grace Bio-Labs. Coverslips were purchased from Thermo Fisher Scientific. The PBST buffer (pH 7.4) used in these experiments contained 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.44 mM KH2PO4, and 0.1% Tween 20. The 1X PBSBT buffer contained 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.44 mM KH2PO4, 0.1% Tween 20, and 1% BSA.
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Preparation of PEG-PCL Nanoparticles for Drug Delivery
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