Biomedical Engineering Reference
In-Depth Information
7.7.1.1 Preparation of the Whole Blood
Dilute the whole blood with DPBS. Expose the blood to acridine orange (AO)
to convert the WBCs nuclei to pinkish yellow color without affecting the RBCs
in order to establish the number of WBCs and RBCs.
(1)
Dilute 10 µL of whole blood at 1:100 with DPBS and mix with the AO at a
ratio of 1:1.
(2)
Incubate the mixture for 10 min at room temperature.
(3)
Centrifuge the cells at 5000 rpm for 5 min.
(4)
Collect the pelleted cells.
(5)
Wash two times with DPBS to remove excess AO.
(6)
Count the stained cells with a hemocytometer under a fluorescence micro-
scope.
7.7.1.2 Capture of the SK-BR3 Cells with IO-Ab
In order to make easy visualization of capture, the freshly harvested SK-BR3
cells are pre-stained with a live cell staining fluorescent dye CMFDA (5-chloro-
methylfluorescein diacetate, Invitrogen, Carlsbad, CA) that converts the cells to
greenish yellow color following the manufacture's staining protocol.
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(1)
Count the cells and spike in 1 mL whole blood sample.
(2)
Add 0.05 mL of IO-Ab NPs at 2 mg Fe/mL.
(3)
Swirl gently to mix at room temperature for 1 h to form IO-Ab + cells.
(4)
Place the mixture in a SuperMag™ separator for 1 h to allow magnetic
isolation of the IO-Ab + cells.
(5)
Withdraw the supernatant gently with a pipettor making sure not to disturb
the captured IO-Ab + cells that stuck on the wall of the tube where the
magnetic field is strongest.
(6)
Resuspend the captured pellet containing the IO-Ab + cells in 100 µL DPBS
for cell inspection and counting.
7.7.1.3 Protocol for the Prussian Blue Staining of the IO-Ab
Captured SK-BR3 Cells
To confirm the IO-Ab tagging of the SK-BR3, the cells were stained with potas-
sium ferrocyanide/HCl mixture.
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The potassium ferrocyanide and HCl mixture
converts the IO from the IO-Ab accumulated on the cells into KFe
III
[Fe
II
(CN)
6
]
which is blue in color.
(1)
Take the SK-BR3 cells grown in a 6-well cell culture plate out of the cell
medium (description of cell culture is described Chapter------).
(2)
Fix the IO-Ab with ice-cold 95% EtOH for 15 min.
(3)
Add the IO-Ab (as 0.5 mg Fe) or IO (as 0.5 mg Fe) NPs in the binding buffer
(20 mm Tris, 150 mm NaCl, 2 mm CaCl
2
, 1 mm MnCl
2
, 1 mm MgCl
2
, 0.1%
(wt/vol) BSA; pH 7.4).
