br sorting analysis To a mg ml solution
sorting) analysis. To a 2 mg/ml solution of PDP-PEG5kDa-SpA in PBS, 1 mM dithiothreitol (DTT) was added and stirred at room temperature for 1 h in order to reduce the disulfide bond of PDP. DTT and pyridine-2-thione were removed by dialysis against degassed PBS plus 5 mM EDTA for 48 h in a nitrogen atmosphere. To a 1.2 mg/ml solution of HS-PEG5kDa-SpA in PBS plus 5 mM EDTA, 2 equivalents of Cy5 maleimide (MW 817 Da) was added and stirred at room temperature overnight in the dark. The reaction was monitored by SEC-HPLC (size exclusion chromatography-HPLC) using a Zorbax GF-250 column (250 × 4.6 mm, Agilent Technologies, Palo Alto, CA) eluted with 20 mM sodium phos-phate, 0.13 M NaCl Cycloheximide 7 with 20% ACN (v/v) at a flow rate of 0.3 ml/ min and measuring the eﬄuent absorbance at 600 nm. The unreacted dye was removed using Pierce™ Dye Removal Columns. The solution was dialyzed against PBS and the dye coupling was quantified by UV–Vis spectrophotometer analysis following the manufacturer’s in-structions.
1 h at room temperature in order to obtain HS-PEG-SpG. DTT and pyridine-2-thione were removed by SEC chromatography using a Superdex® 200 Increase 10/300 GL column (30 cm × 10 mm, 8.6 μm particle size, GE Healthcare) eluting with PBS and 5 mM EDTA pH 7.4 at 0.5 ml/min and measuring absorbance at 280 nm. To the purified peak of HS-PEG-SpG, 3 equivalent of Cy5-maleimide was added. The reactions were allowed to proceed in the dark overnight at room tem-perature. Unreacted dye was removed using Pierce™ Dye Removal Columns and dye removal was confirmed by SEC-HPLC. The solution was dialyzed against PBS, and dye coupling was quantified by UV–Vis spectrophotometer analysis following the manufacturer’s instructions.
A 5–10 fold molar excess of Cy5-NHS (MW 791.99 Da) was added to a 1–2 mg/ml solution of a mAb (Trz, Rtx and Bevacizumab) in PBS, and the mixture was stirred in the dark overnight at room temperature. After the unreacted dye was removed with Pierce™ Dye Removal Columns, mAbs concentration and dye loading were calculated by UV–Vis spectroscopy following the manufacturer’s instructions.
The following human tumor cell lines were used: BL-41, a Burkitt lymphoma B cell line; Raji, a lymphoblast-like cell line derived from a Burkitt lymphoma; LCL, a lymphoblastoid cell line generated by Epstein-Barr virus (EBV) infection of peripheral blood mononuclear cell (PBMC); Jurkat, a T cell lymphoma cell line; IGROV-1 and SKOV-3, which are ovarian adenocarcinoma cell lines; MDA-MB-231 and SK-BR-3, which are breast cancer cell lines.
The cells were grown in RPMI 1640 (except for MDA-MB-231 and SK-BR-3, which were cultured in McCoy’s 5A and DMEM, respectively) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 200 U/ml penicillin, 200 U/ml streptomycin and 1 mM sodium pyruvate, hereafter referred as to complete medium.
The cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
2.9. Cytometry analysis
CD20 expression in BL-4, Raji, LCL, and Jurkat cell lines, and HER2/ neu expression in IGROV-1, SKOV-3, MDA/MB-231, and SK-BR-3 were evaluated by flow cytometry. The cells (3 × 105/sample) were re-suspended in 50 μl fluorescence-activated cell sorter (FACS) buﬀer
(0.9% NaCl solution containing 2% bovine serum albumin and 0.02% NaN3), and stained at 4 °C for 30 min with PE-conjugated mouse anti-human CD20 mAb or with Trz (1:100), and then for another 30 min at 4 °C with PE-conjugated mouse anti-human IgG1 mAb. Before they were analyzed, the cells were washed twice, resuspended in FACS buﬀer, and analyzed with a flow cytometer FACS-CALIBUR (Becton Dickinson, Erembodegem, Belgium). Data analysis was performed using the FlowJo 7.6.5 data analysis software package (TreeStar, USA).