Dspe peg 2000 maleimide

A 117 amino-acid peptide corresponding to RH5Nt (F1-K116) with an additional C-terminal cysteine was chemically synthesized (LifeTein). The peptide was dissolved in DMF and conjugated to maleimide-PEG2000-DSPE (Avanti Polar Lipids) at a 1:2 molar ratio overnight at room temperature with rotation. Polyclonal antisera were raised by immunizing rabbits six times with 100 μg of purified antigen in Freund's adjuvant in rabbits (Cambridge Research Biochemicals) in strict accordance with UK Home Office governmental regulations and the local Sanger Institute animal welfare ethical review board. Polyclonal antisera were similarly raised against full-length CyRPA and the P113 Y1-N653 fragment expressed in mammalian cells as rat Cd4d3+4-6His tagged proteins and purified using Ni²⁺-NTA chromatography. The sera were purified according to manufacturer's instructions on an ÄKTA system using separate Hi-Trap Protein G HP columns (GE Healthcare) for each rabbit serum. The fractions were eluted with 0.2 M glycine (pH 2.7) directly into a neutralization buffer (1 M Tris:Cl pH 9.0) and immediately dialysed into PBS.

Paclitaxel (purity > 99.9%) was purchased from Zelang Medical Technology Co., Ltd. (Nanjing, China). DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide) (purity > 95.0%), Dithiodiglycolic acid (purity > 96.0%), 1-Octadecanol (purity > 99.0%), Glutathione(purity > 98.0%) and were obtained from Aladdin Reagent Database Inc. (Shanghai, China). CGKRK peptides (purity > 95%) were purchased from GL Biochem Co., Ltd. (Shanghai, China), Maleimide-PEG2000-DSPE (purity > 99%) and MeO-PEG2000-DSPE (purity > 99%) were purchased from Avanti Polar Lipids, Inc. (Alabama, USA). Bor (purity > 97.0%), glutathione-reduced ethyl ester (purity > 90.0%), and Coumarin-6 (purity ≥ 99%) were provided by Sigma-Aldrich (St. Louis, MO, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (purity >99%) and BCA kit were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). Penicillin-streptomycin, DMEM medium, fetal bovine serum (FBS), and 0.25% (w/v) trypsin solution were purchased from Gibco BRL (MD, USA). The other chemical reagents were of analytical grade and used as received.

All starting materials were purchased from Sigma Aldrich (St. Louis, MO, USA), unless otherwise noted. DSPE-PEG₂₀₀₀-maleimide was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cy7-amine was purchased from the Lumiprobe corporation (Hunt Valley, MD, USA), and Cy7 mono-N-hydroxysuccinimide ester was purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Materials for cell culture, which include fetal bovine serum (FBS, Gibco, Gaithersburg, MD USA), penicillin-streptomycin (Gibco, USA), 2-mercaptoethanol (Gibco, USA), and PBS (Gibco, USA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Sigma Aldrich. WEHI-274.1 murine monocytes (ATCC# CRL-1679) were purchased from ATCC (Manassas, VA, USA) and subjected to mycoplasma testing.