2.4 Cell Culture
Maintenance
1. U-118 MG cell line (ATCC).
2. Dulbecco’s Modified Eagle Medium (DMEM) High Glucose.
3. Fetal bovine serum (FBS).
4. Penicillin-streptomycin.
5. Trypsin.
6. Phosphate-buffered saline (PBS).
7. T-75 flasks.
8. CO 2 incubator.
2.5 Visualization of
Transfection
Complexes in the Cells
1. Atto 550 NHS solution (10 mg/mL): 1 mg of Atto 550 NHS,
100 μL of DMSO. Store at À20
C.
2. Borate buffer: 0.1 M borate, pH 8.5, 1 M NaOH in H 2 O.
3. Slide-A-Lyzer G2 Dialysis Cassettes, 3.5 k MWCO, 3 mL
(Thermo Fisher Scientific).
4. 8-well Nunc Lab-Tek Chamber Slide (Thermo Fisher
Scientific).
5. Label IT Tracker Intracellular Nucleic Acid Localization Kit,
Fluorescein (Mirus).
6. 4% formaldehyde solution: 1 mL of 16% formaldehyde, 3 mL
of Dulbecco’s phosphate-buffered saline containing MgCl 2
and CaCl 2 (DPBS).
7. Concanavalin A-FITC (Thermo Fischer) solution (1 mg/mL):
10 mg of Concanavalin A-FITC, 10 mL of 0.1 M sodium
bicarbonate. Aliquot and store at À20
C.
2.6 Cytotoxicity
Assays
1. Black polystyrene 96-well flat bottom μClear plate with the
transparent bottom (Greiner Bio-One GmbH).
2. Live/Dead assay solution: 2 μM calcein AM, 2 μM ethidium
homodimer-1, 8 μM Hoechst 33342 in DPBS. Protect from
light, storing in a tube wrapped with aluminum foil.
2.7 Transfection
1. DMEM High Glucose.
2. PBS.
3. 12-well cell culture plate.
2.8 Establishing
TNC-C
Expression Level
2.8.1 Total RNA Isolation
from Cell Culture
1. TRIzol Reagent.
2. Chloroform.
3. 2-Propanol.
4. 75% ethanol.
5. Refrigerated microfuge.
6. Microvolume spectrophotometer.
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Małgorzata Grabowska et al.
Maintenance
1. U-118 MG cell line (ATCC).
2. Dulbecco’s Modified Eagle Medium (DMEM) High Glucose.
3. Fetal bovine serum (FBS).
4. Penicillin-streptomycin.
5. Trypsin.
6. Phosphate-buffered saline (PBS).
7. T-75 flasks.
8. CO 2 incubator.
2.5 Visualization of
Transfection
Complexes in the Cells
1. Atto 550 NHS solution (10 mg/mL): 1 mg of Atto 550 NHS,
100 μL of DMSO. Store at À20
C.
2. Borate buffer: 0.1 M borate, pH 8.5, 1 M NaOH in H 2 O.
3. Slide-A-Lyzer G2 Dialysis Cassettes, 3.5 k MWCO, 3 mL
(Thermo Fisher Scientific).
4. 8-well Nunc Lab-Tek Chamber Slide (Thermo Fisher
Scientific).
5. Label IT Tracker Intracellular Nucleic Acid Localization Kit,
Fluorescein (Mirus).
6. 4% formaldehyde solution: 1 mL of 16% formaldehyde, 3 mL
of Dulbecco’s phosphate-buffered saline containing MgCl 2
and CaCl 2 (DPBS).
7. Concanavalin A-FITC (Thermo Fischer) solution (1 mg/mL):
10 mg of Concanavalin A-FITC, 10 mL of 0.1 M sodium
bicarbonate. Aliquot and store at À20
C.
2.6 Cytotoxicity
Assays
1. Black polystyrene 96-well flat bottom μClear plate with the
transparent bottom (Greiner Bio-One GmbH).
2. Live/Dead assay solution: 2 μM calcein AM, 2 μM ethidium
homodimer-1, 8 μM Hoechst 33342 in DPBS. Protect from
light, storing in a tube wrapped with aluminum foil.
2.7 Transfection
1. DMEM High Glucose.
2. PBS.
3. 12-well cell culture plate.
2.8 Establishing
TNC-C
Expression Level
2.8.1 Total RNA Isolation
from Cell Culture
1. TRIzol Reagent.
2. Chloroform.
3. 2-Propanol.
4. 75% ethanol.
5. Refrigerated microfuge.
6. Microvolume spectrophotometer.
72
Małgorzata Grabowska et al.