acquired on the Olympus FV1000 with a Â60 objective, a 1.4
oil immersion lens, and analyzed using the FV10-ASW software (see Note 5).
3.6 Cytotoxicity
Assays
3.6.1 WST-1 Cell
Proliferation Assay
1. Seed U-118 MG cells into 96-well plate at 1 Â 10
4 cells/well
(150 μL) and incubate under standard tissue culture conditions
for 24 h.
2. Prepare dilutions of transfection complexes with an iron-toRNA ratio of 3:1 (w/w) corresponding to the ATN-RNA final
concentration of 100, 50, 25, 12.5, and 6.25 nM. Transfer
50 μL each of the transfection complexes into the culture plates
with the seeded cells. Incubate the cells with complexes for
24 h. Test each dilution of transfection complex in triplicate.
3. Add 10 μL WST-1 Cell Proliferation Reagent to each well and
incubate at 37
C for 4 h. Use cell culture medium as a blank.
4. Transfer 100 μL medium to the fresh wells on 96-well plate and
record the absorbance at 450 nm (reference wavelength
620 nm) using a multiwell plate reader. Use untransfected
cells as a reference.
5. Express the cell viability as: Cell viability (%) ¼ (Abs sample –
Abs blank )/(Abs ref – Abs blank ) * 100. Abs sample , Abs blank , and
Abs ref are absorbances at the maximum of the WST-1 absorption spectrum registered for a sample, blank, and reference
sample, respectively.
3.6.2 Live/Dead Cell
Viability Assay
1. Seed U-118 MG cells into 96-well μClear plate at
1 Â 10
4 cells/well (150 μL) and incubate under standard tissue
culture conditions for 24 h.
2. Prepare dilutions of transfection complexes with an iron-toRNA ratio of 3:1 (w/w) corresponding to the ATN-RNA final
concentration of 100, 50, 25, 12.5, and 6.25 nM. Transfer
50 μL each of the transfection complexes into the culture plates
with the seeded cells. Incubate the cells with complexes for
24 h. Test each dilution of transfection complex in triplicate.
3. Remove medium from all wells and replace with 100 μL of
Live/Dead assay solution. Incubate at 37
C for 30 min.
4. Scan the plate using a high-content imaging system. For the
experiments detailed in this section, the images were acquired
on the IN Cell Analyzer 2000 with a Â20 objective and analyzed using the IN Cell Developer Toolbox software using
in-house developed protocol (see Note 6).
3.7 Transfection
1. Calculate the volumes of nanoparticles and dsRNA needed for
transfection based on binding efficiency obtained from Subheadings 3.3.1 and 3.3.2. Determine what volume of dsRNA
Delivery of dsRNA to Glioblastoma Cells
77
oil immersion lens, and analyzed using the FV10-ASW software (see Note 5).
3.6 Cytotoxicity
Assays
3.6.1 WST-1 Cell
Proliferation Assay
1. Seed U-118 MG cells into 96-well plate at 1 Â 10
4 cells/well
(150 μL) and incubate under standard tissue culture conditions
for 24 h.
2. Prepare dilutions of transfection complexes with an iron-toRNA ratio of 3:1 (w/w) corresponding to the ATN-RNA final
concentration of 100, 50, 25, 12.5, and 6.25 nM. Transfer
50 μL each of the transfection complexes into the culture plates
with the seeded cells. Incubate the cells with complexes for
24 h. Test each dilution of transfection complex in triplicate.
3. Add 10 μL WST-1 Cell Proliferation Reagent to each well and
incubate at 37
C for 4 h. Use cell culture medium as a blank.
4. Transfer 100 μL medium to the fresh wells on 96-well plate and
record the absorbance at 450 nm (reference wavelength
620 nm) using a multiwell plate reader. Use untransfected
cells as a reference.
5. Express the cell viability as: Cell viability (%) ¼ (Abs sample –
Abs blank )/(Abs ref – Abs blank ) * 100. Abs sample , Abs blank , and
Abs ref are absorbances at the maximum of the WST-1 absorption spectrum registered for a sample, blank, and reference
sample, respectively.
3.6.2 Live/Dead Cell
Viability Assay
1. Seed U-118 MG cells into 96-well μClear plate at
1 Â 10
4 cells/well (150 μL) and incubate under standard tissue
culture conditions for 24 h.
2. Prepare dilutions of transfection complexes with an iron-toRNA ratio of 3:1 (w/w) corresponding to the ATN-RNA final
concentration of 100, 50, 25, 12.5, and 6.25 nM. Transfer
50 μL each of the transfection complexes into the culture plates
with the seeded cells. Incubate the cells with complexes for
24 h. Test each dilution of transfection complex in triplicate.
3. Remove medium from all wells and replace with 100 μL of
Live/Dead assay solution. Incubate at 37
C for 30 min.
4. Scan the plate using a high-content imaging system. For the
experiments detailed in this section, the images were acquired
on the IN Cell Analyzer 2000 with a Â20 objective and analyzed using the IN Cell Developer Toolbox software using
in-house developed protocol (see Note 6).
3.7 Transfection
1. Calculate the volumes of nanoparticles and dsRNA needed for
transfection based on binding efficiency obtained from Subheadings 3.3.1 and 3.3.2. Determine what volume of dsRNA
Delivery of dsRNA to Glioblastoma Cells
77