93
12. Remove MTT solution.
13. Solubilize the formazan crystals by the addition of 100 μL
DMSO or isopropanol.
14. Record the absorbance using a UV-vis microplate reader at a
specific wavelength (see Note 5).
15. Average control wells’ absorption readings and calculate standard deviation.
16. Average the treated triplicate wells’ absorption readings and
standard deviation (SD).
17. Subtract control average from each treated average absorption
value.
18. Convert to percentile values that express cellular proliferation,
aka cell viability (see Note 6).
19. Plot the % viability (y-axis) versus the tested sample concentrations (x-axis) in a column graph expressing the SD with error
bars.
The induced cytotoxicity when cells are treated with mNCs drug
loaded or unloaded can be assessed by performing the MTT assay
after the application of magnetic hyperthermia in treated cell samples. According to our protocol the steps are briefly described
below:
1. Treat the appropriate number of cells with mnps or magnetic
NCs in desired concentrations.
2. Incubate the cells for 24 h (see Subheading 3.2, step 2).
3. Wash with PBS twice to remove the non-internalized
material.
4. Add the appropriate amount of trypsin to suspend the cells.
5. Suspend cells to growth medium in the appropriate concentration in sterile tube.
6. Treat the solution with alternating magnetic field for 30 min
in agreement to hyperthermia measurements ex vitro.
7. Seed the treated cells of different concentrations of drugloaded and -unloaded mNCs or mNPs in a 96-well plate.
8. Apply MTT assay (see Subheading 3.2, step 2).
9. In this way induced cytotoxicity from the treated samples can
be indirectly calculated from the % viability.
The scratch-wound healing assay [23] is performed usually in
6-,12-, or 24-well plates with plastic or glass slides in each well,
where the cells are seeded in the appropriate growth medium
(Fig. 2). The same number of cells should be seeded (3 × 10^5
cell/mL) in each well in order to have comparable cell culture
monolayers. As soon as confluency is reached (approx. 24 h) the
assay can begin:
3.3.3 In Vitro Cytotoxicity
Studies Under
Hyperthermia
3.4 Scratch-Wound
Healing
Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation
12. Remove MTT solution.
13. Solubilize the formazan crystals by the addition of 100 μL
DMSO or isopropanol.
14. Record the absorbance using a UV-vis microplate reader at a
specific wavelength (see Note 5).
15. Average control wells’ absorption readings and calculate standard deviation.
16. Average the treated triplicate wells’ absorption readings and
standard deviation (SD).
17. Subtract control average from each treated average absorption
value.
18. Convert to percentile values that express cellular proliferation,
aka cell viability (see Note 6).
19. Plot the % viability (y-axis) versus the tested sample concentrations (x-axis) in a column graph expressing the SD with error
bars.
The induced cytotoxicity when cells are treated with mNCs drug
loaded or unloaded can be assessed by performing the MTT assay
after the application of magnetic hyperthermia in treated cell samples. According to our protocol the steps are briefly described
below:
1. Treat the appropriate number of cells with mnps or magnetic
NCs in desired concentrations.
2. Incubate the cells for 24 h (see Subheading 3.2, step 2).
3. Wash with PBS twice to remove the non-internalized
material.
4. Add the appropriate amount of trypsin to suspend the cells.
5. Suspend cells to growth medium in the appropriate concentration in sterile tube.
6. Treat the solution with alternating magnetic field for 30 min
in agreement to hyperthermia measurements ex vitro.
7. Seed the treated cells of different concentrations of drugloaded and -unloaded mNCs or mNPs in a 96-well plate.
8. Apply MTT assay (see Subheading 3.2, step 2).
9. In this way induced cytotoxicity from the treated samples can
be indirectly calculated from the % viability.
The scratch-wound healing assay [23] is performed usually in
6-,12-, or 24-well plates with plastic or glass slides in each well,
where the cells are seeded in the appropriate growth medium
(Fig. 2). The same number of cells should be seeded (3 × 10^5
cell/mL) in each well in order to have comparable cell culture
monolayers. As soon as confluency is reached (approx. 24 h) the
assay can begin:
3.3.3 In Vitro Cytotoxicity
Studies Under
Hyperthermia
3.4 Scratch-Wound
Healing
Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation