Biomedical Engineering Reference
In-Depth Information
8.2.4.1 Procedure for In vitro Evaluation of IOMNP Toxicity using
the MTT Assay
131,132
The effect of amphiphilic polymer coated water-soluble IOMNPs at various
concentrations to the growth of the cell line U937 was investigated.
132
The IOMNPs in the study were 5 and 10 nm in diameter.
(1)
Grow the U937 cells from newly harvested suspension in culture flasks
following the manufacturer's recommendation.
(2)
Harvest the cells when they reach about 70-80% confluence.
(3)
Place 10
5
cells from the newly harvested suspension in individual centri-
fuge tubes.
(4)
Add enough IOMNPs to each tube to attain the final concentrations of
0.025, 0.5, and 2 µM aqueous solutions using the medium as diluent.
(5)
Incubate at 37 °C in 5% CO
2
for 1, 4, and 24 h.
(6)
Spin the cells down at 3000 rpm for 3-4 min to collect the cells.
(7)
Wash the cells three times with 1× DPBS to remove NPs.
(8)
Resuspend the cells in 1× DPBS and place in a 96-well plate.
(9)
Perform MTT assay following the manufacturer's recommendation.
(10)
Dissolve the crystals formed in 50 uL of MTT buffer.
(11)
Scan the solutions at 595 nm and subtract the background signal.
The MTT assay evaluation of IOMNP toxicity did not exhibit toxic effects of
both the 5 nm and 10 nm diameter particles that were coated with amphiphilic
polymer
131
(
Table 8.5
). These results are important for the use of IOMNPs in
animal studies for evaluations of their medical applications. However, because
of the difficulty in reading the signals resulting from the presence of NMs in the
cells that may have affected the results of the study, a more direct approach to cell
death was used as an alternative method to MTT. This process is given below.
8.2.4.2 Procedure for the Evaluation of IOMNPs Toxicity to U937
Cells using the DAPI Staining and Cell Count Method
In order to have a better idea of the toxicity of IOMNP on cells, an alternative
strategy that involves actual counting of cells, both viable and non-viable, can
be used. Using DAPI stain, the dead cells absorb the dye whereas live cells do
not. Briefly, the assay is performed as follows.
(1)
Grow the cells on glass cover slip inserted in a 6-well plate until they are
30-50% confluent.
(2)
Replace the medium with a new medium to which the IOMNPs are mixed
to attain the desired concentrations.
(3)
Incubate the plates for 24 h at 37 °C and 5% CO
2
.
(4)
Remove the medium with NPs.
(5)
Add 1 mL of 1 × DPBS to each well to wash the cells.
(6)
Repeat the washing 2 more times.
(7)
Add 1mL of 1 × DPBS to each well.
