6. Wash the cells 1Â with DPBS and then fix the cells by adding
200 μL of 4% formaldehyde and incubate for 15 min at room
temperature.
7. Wash the cells 1Â with DPBS and label the cellular membranes
by adding 200 μL of Concanavalin A-FITC at the concentration of 25 μg/mL and incubate for 30 min at room temperature. Protect from light.
8. Wash the cells 1Â with DPBS and label the nuclei by adding
200 μL of Hoechst 33342 at the concentration of 8 μM and
incubate for 10 min at room temperature. Protect from light.
9. Wash the cells 1Â with DPBS. Samples may be stored in 300 μL
at 4
C.
10. Visualize the cells using confocal laser scanning microscope.
For the experiments detailed in this section, the images were
acquired on the Olympus FV1000 with a Â60 objective, a 1.4
oil immersion lens, and analyzed using the FV10-ASW software (see Note 4).
3.5.2 Colocalization of
Magnetic Nanoparticles
and RNA Inside the Cells
1. To obtain fluorescently labeled ATN-RNA with Label IT
Tracker Intracellular Nucleic Acid Localization Kit, mix 5 μg
of RNA with 10Â Labeling Buffer A, Label IT
® Reagent and
DNase-, RNase-free water in a volume of 50 μL.
2. Quantify the final concentration on a spectrophotometer.
3. Seed U-118 MG cells into 8-well Nunc Lab-Tek Chamber
Slide at 2.5 Â 10
4 cells/well (300 μL) and incubate under
standard tissue culture conditions for 24 h.
4. Prepare working dilution of transfection complexes containing
Atto 550 NHS-labeled NPs (red) and fluorescein-labeled RNA
(green) with an iron-to-RNA ratio of 3:1 (w/w). Transfer
50 μL of the transfection complexes corresponding to the
ATN-RNA final concentration of 100 nM into the well of
slide with the seeded cells. Incubate the cells with complexes
for 24 h.
5. Wash the cells 1Â with DPBS and then fix the cells by adding
200 μL of 4% formaldehyde and incubate for 15 min at room
temperature. Protect from light.
6. Remove fixative from the cells and then wash the cells 1Â
with DPBS.
7. Label the nuclei by adding 200 μL of Hoechst 33342 at the
concentration of 8 μM and incubate for 10 min at room
temperature. Protect from light. Samples may be stored in
300 μL at 4
C.
8. Visualize the cells using confocal laser scanning microscope.
For the experiments detailed in this section, the images were
76
Małgorzata Grabowska et al.
200 μL of 4% formaldehyde and incubate for 15 min at room
temperature.
7. Wash the cells 1Â with DPBS and label the cellular membranes
by adding 200 μL of Concanavalin A-FITC at the concentration of 25 μg/mL and incubate for 30 min at room temperature. Protect from light.
8. Wash the cells 1Â with DPBS and label the nuclei by adding
200 μL of Hoechst 33342 at the concentration of 8 μM and
incubate for 10 min at room temperature. Protect from light.
9. Wash the cells 1Â with DPBS. Samples may be stored in 300 μL
at 4
C.
10. Visualize the cells using confocal laser scanning microscope.
For the experiments detailed in this section, the images were
acquired on the Olympus FV1000 with a Â60 objective, a 1.4
oil immersion lens, and analyzed using the FV10-ASW software (see Note 4).
3.5.2 Colocalization of
Magnetic Nanoparticles
and RNA Inside the Cells
1. To obtain fluorescently labeled ATN-RNA with Label IT
Tracker Intracellular Nucleic Acid Localization Kit, mix 5 μg
of RNA with 10Â Labeling Buffer A, Label IT
® Reagent and
DNase-, RNase-free water in a volume of 50 μL.
2. Quantify the final concentration on a spectrophotometer.
3. Seed U-118 MG cells into 8-well Nunc Lab-Tek Chamber
Slide at 2.5 Â 10
4 cells/well (300 μL) and incubate under
standard tissue culture conditions for 24 h.
4. Prepare working dilution of transfection complexes containing
Atto 550 NHS-labeled NPs (red) and fluorescein-labeled RNA
(green) with an iron-to-RNA ratio of 3:1 (w/w). Transfer
50 μL of the transfection complexes corresponding to the
ATN-RNA final concentration of 100 nM into the well of
slide with the seeded cells. Incubate the cells with complexes
for 24 h.
5. Wash the cells 1Â with DPBS and then fix the cells by adding
200 μL of 4% formaldehyde and incubate for 15 min at room
temperature. Protect from light.
6. Remove fixative from the cells and then wash the cells 1Â
with DPBS.
7. Label the nuclei by adding 200 μL of Hoechst 33342 at the
concentration of 8 μM and incubate for 10 min at room
temperature. Protect from light. Samples may be stored in
300 μL at 4
C.
8. Visualize the cells using confocal laser scanning microscope.
For the experiments detailed in this section, the images were
76
Małgorzata Grabowska et al.