Nhs fitc

Indirect and direct methods were used to test the cellular affinity according to previous protocols.^[19] Briefly, for indirect method, lymphoma cells were incubated with dara (5 × 10⁻⁹m) and then a goat anti‐human secondary antibody (3 µg mL⁻¹, ThermoFisher Scientific) in Flow Cytometry Staining Buffer Solution (eBioscience) at the concentration of 1 × 10⁶ cells mL⁻¹. For direct method,
N‐Hydroxysuccinimide –fluorescein (NHS–FITC, ThermoFisher)‐conjugated antibody was prepared and purified.^[25] Then, FITC‐conjugated DTPA– or Df–dara were tested and processed using a LSRFortessa cell analyzer (BD Biosciences) and FlowJo software (Tree Star) for the mean fluorescence intensities.

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-ethyldiisopropylamine (N,N-diisopropylethlamine) (DIEA, 99%), and thiazolyl blue tetrazolium bromide (MTT, 98%) were from Alfa Aesar (Echo Chemical Co., Ltd., Heysham, UK). N-hydroxysuccinimide (NHS, 98%) and poly(vinyl alcohol) (PVA, 88% hydrolyzed, 20,000–30,000 g/mol) were from Acros Organics Co., Inc. (Fair Lawn, NJ, USA). Poly(d,l-lactide-co-glycolide) 50:50 (PLGA, ~52,000 g/mol) was from Evonik Industries (Birmingham, AL, USA). Maleimide poly(ethylene glycol) amine (Mal-PEG-amine, 5000 g/mol) was provided by Hunan Hua Teng Pharmaceutical Co., Ltd. (Merelbeke, Belgium). FITC-NHS (MW 473.4 g/mol) was from Thermo Fisher Scientific Inc. (Hudson, NH, USA). FITC-Cys-T7 peptide (1498.71 g/mol, FITC-Cys-His-Ala-Ile-Tyr-Pro-Arg-His-OH) was from Kelowna International Scientific Inc. (Taipei, Taiwan). Anti-human CD71 (transferrin receptor) monoclonal antibody, allophycocyanin (APC) and mouse IgG1 kappa Isotype Control APC were from eBioscience, Inc. (Vienna, Austria). Seliciclib (purity > 99%) was from LC Laboratories (Woburn, MA, USA). A549, MDA-MB-231, SKOV-3 and U87-MG cell lines were from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Recombinant antibodies were generated using the Expi293 (ThermoFisher) or Expi293 FUT8^−/− system using previously described protocols (47 (link)). Briefly, an equal ratio of heavy- and light-chain plasmids was complexed with ExpiFectamine in OptiMEM and added to Expi293 cells in culture at 3 × 10⁶ cells/ml. Enhancer 1 and Enhancer 2 were added 20 h after transfection. After 6 d, recombinant IgG antibodies were purified from cell-free supernatants by affinity purification using protein G sepharose beads (GE Healthcare), dialyzed in PBS, filter-sterilized (0.22 μm), concentrated with 100 kDa MWCO spin concentrator (Millipore), purified with Superdex 200 Increase 10/300 GL (GE Healthcare), and finally assessed by SDS–PAGE followed by SafeBlue staining (ThermoFisher). All antibody preparations were more than 95% pure and endotoxin levels were less than 0.05 EU mg⁻¹, as measured by the Limulus amebocyte lysate assay. Purified IgG was fluorescently labeled with Alexa Fluor 647-NHS or FITC-NHS (ThermoFisher) at a 15-fold molar excess for 1 h at room temperature and double-dialyzed into PBS.