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Scheme 1. Schematic representation of antibody-conjugated MNP-Si preparation. (A), Separation of Sk-BR-3 cells from PBMCs with Ab/MNP-Si. (B) Analysis with flow cytometry (C).
washed with PBS 1× in a magnetic field (gradient of 0.5 T/m) for 15–20 min. Because the final product (Ab/MNP-Si) has paramagnetic properties, it sediments on the magnet and the rest of the mixture can be separated and removed. This process was repeated two more times. Then the Herceptin-conjugated MNP-Si was dispersed in storage buﬀer (BPS 1× containing 0.1% FBS, 0.05% sodium azide) at a final con-centration of 0.9 mg/mL.
2.4. Flow cytometric analysis of antibody-conjugated MNPs
To ensure that the conjugation process was complete, Herceptin binding to MNP-Si was verified with flow cytometry. For this purpose, Ab/MNP-Si was first ultrasonicated on ice for 1 min at 15-20 KHz (10 s oﬀ and 10 s on) to ensure complete dispersion. A volume of 40 µL so-nicated Ab/MNP-Si was then poured into a flow cytometry tube and stained with FITC-conjugated mouse anti-human IgG1 and leftin the dark for 20 min at room temperature. Simultaneously, a tube containing MNP-Si without Herceptin was used as a negative control. All tubes were then washed with 2 mL PBS 1× (300 × g/5 min) and tested with flow cytometry.
Cells from the HER2-positive human BC cell line SK-BR-3were cul-tured in complete culture medium containing RPMI-1640 supple-mented with 20% heat-inactivated FBS and 1% penicillin/strepto-mycin, and kept in a humidified Ursodeoxycholylglycine with 5% CO2 at 37 °C in a cell culture incubator. When 80–90% confluency was obtained, the cells were trypsinized and harvested, washed and resuspended in fresh medium. Then trypan blue staining was used to count the cells in a hemocytometer under an inverted microscope to determine cell viabi-lity.
2.6. Cancer cell separation with Ab/MNP-Si from peripheral blood mononuclear cells
The ability of Ab/MNP-Si to attach to SK-BR-3 cells was checked with a flow cytometry-based method. For this purpose, diﬀerent ratios of SK-BR-3 cells and peripheral blood mononuclear cells (PBMCs) were mixed. The number of PBMCs was kept at 1.7 million in all tubes, and these cells were mixed with diﬀerent numbers of SK-BR-3 cells (0.1– 4 × 105). Then, 25 µL ultrasonicated Ab/MNP-Si and 2 µL DNase were added and the mixture was stirred for 2 h. After reaction, PBMCs and
Fig. 7. Flow cytometric diagrams of MNPs coated with silane (MNP-Si) with and without Herceptin. MNPs conjugated without (a) and with (b) Herceptin were stained with FITC mouse anti-human IgG1 and gated according to their forward and side scatters. The purple areas show MNP-Si without Herceptin (c) and the blue curve shows Herceptin-conjugated MNP-Si (d). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
unreacted SK-BR-3 cells were washed 3 times with PBS 1× in a mag-netic field for 15 min. Simultaneously, for each reaction, similar tubes with the same numbers of PBMC, Ab/MNP-Si and SK-BR-3 cells were used as controls. The control tubes underwent no washing and no se-dimenting in a magnetic field.
The isolated cells and the cells in the control tube were then stained with 10 µL PE-conjugated EpCAM and FITC-conjugated CD45, and in-cubated in the dark for 20 min at room temperature. The tubes were then washed with 2 mL PBS 1× (650 g for 5 min) and stained with 10 µL 7-AAD. After 20 min of incubation in the dark at room tem-perature, 300 µL PBS 1× was added to all tubes, which were then evaluated with flow cytometry.
2.7. Cancer cell separation using Ab/MNP-Si in a mixture of fresh human whole blood
To analyze the ability of Ab/MNP-Si to isolate HER2+ cells, SK-BR-3 cells were stained with carboxyfluorescein succinimidyl ester (CFSE) as a live cell-staining fluorescent dye (which changes the color of the cells to green) according to the manufacturer’s protocol. Stained SK-BR-3 cells were then mixed with 1 mL fresh human whole blood. A volume of 90 µL Ab/MNP-Si was added to the SK-BR-3/whole blood mixture and stirred at room temperature for 1 h. The mixture was then placed on a magnet for 20 min to allow magnetic isolation of the Ab/MNP-Si attached cells. The captured Ab/MNP-Si were attached on the tube wall, and the supernatant fluid was discarded. The pellet containing
Ab/MNP-Si was resuspended in 100 µL PBS 1× for cell detection with fluorescence microscopy, except for the experiment with 100 SK-BR-3 cells, where captured Ab/MNP-Si were detected directly with fluores-cence microscopy.
3.1. Checking MNP characteristics