92
metal complexes. The assay is based on enzymatic reduction of
3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) to MTT-formazan which is catalyzed by mitochondrial
succinate dehydrogenase. MTT assay is dependent on mitochondrial respiration and indirectly assesses the cellular energy capacity
of a cell [22]. Cytotoxicity of the NCs can be evaluated by the
MTT assay on different human cell lines.
In order to perform the MTT assay (Fig. 1) the protocol is as
follows:
Following step 8 of the cell culture procedure (Subheading
3.3.1) described previously,
1. Seed 100 μL of 8 × 10
3
cells in each well in 96-well plate, flatbottomed microplates.
2. Incubate the cells 24 h before the experiment.
3. Disperse the NCs/mNCs, free drug, and loaded NCs/mNCs
in ddH 2 O or growth medium as stock solution.
4. Create serial dilutions of comparable concentration for each
sample in growth medium of at least six different
concentrations.
5. After 24 h (70% confluency), remove supernatant growth
medium.
6. Remove cellular debris by adding and quickly but gently
removing 100 μL PBS.
7. Add 100 μL of each concentration in row triplicate wells as
well as 3 columns of control wells in each plate containing
pure growth medium.
8. Incubate the cells for different time intervals (e.g., 4, 24, 48,
and 72 h).
9. Remove the supernatant and wash twice with PBS.
10. Replace with 100 μL MTT solution.
11. Incubate for 4 h, or until the formazan crystals have formed.
Fig. 1 Schematic representation of MTT assay procedure
Maria Theodosiou et al.
metal complexes. The assay is based on enzymatic reduction of
3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) to MTT-formazan which is catalyzed by mitochondrial
succinate dehydrogenase. MTT assay is dependent on mitochondrial respiration and indirectly assesses the cellular energy capacity
of a cell [22]. Cytotoxicity of the NCs can be evaluated by the
MTT assay on different human cell lines.
In order to perform the MTT assay (Fig. 1) the protocol is as
follows:
Following step 8 of the cell culture procedure (Subheading
3.3.1) described previously,
1. Seed 100 μL of 8 × 10
3
cells in each well in 96-well plate, flatbottomed microplates.
2. Incubate the cells 24 h before the experiment.
3. Disperse the NCs/mNCs, free drug, and loaded NCs/mNCs
in ddH 2 O or growth medium as stock solution.
4. Create serial dilutions of comparable concentration for each
sample in growth medium of at least six different
concentrations.
5. After 24 h (70% confluency), remove supernatant growth
medium.
6. Remove cellular debris by adding and quickly but gently
removing 100 μL PBS.
7. Add 100 μL of each concentration in row triplicate wells as
well as 3 columns of control wells in each plate containing
pure growth medium.
8. Incubate the cells for different time intervals (e.g., 4, 24, 48,
and 72 h).
9. Remove the supernatant and wash twice with PBS.
10. Replace with 100 μL MTT solution.
11. Incubate for 4 h, or until the formazan crystals have formed.
Fig. 1 Schematic representation of MTT assay procedure
Maria Theodosiou et al.