2.5 Cell Culture
1. HEK293 cells (American Type Culture Collection) (see
Note 8).
2. Cell culture medium: Dulbecco’s modified Eagle’s medium
(DMEM) without phenol red and antibiotics, supplemented
with 10% (v/v) fetal bovine serum (FBS) and 2 mM Lglutamine.
3. Phosphate-buffered saline (PBS): 0.2 g/L KCl, 0.24 g/L
KH 2 PO 4 , 8 g/L NaCl, and 1.44 g/L Na 2 HPO 4 (anhydrous)
in nuclease-free water.
4. Trypsin: Phenol red-free solution of 0.25% (w/v) trypsin
diluted in PBS.
5. 6-Well plates.
2.6 Microporation
(See Note 9)
1. Microporator.
2. PBS.
3. Electroporation buffer (see Note 9).
4. Electrolytic buffer (see Note 9).
5. Electroporation chamber (see Note 9).
6. Electroporation tube (see Note 9).
7. Cell culture medium (as in Subheading 2.5, see Note 10).
8. 8-Well chambered cover glass.
9. 10 μg/mL Fibronectin.
10. Refrigerated microcentrifuge.
11. Cell-counting device.
2.7 Fluorescence In
Situ Hybridization
(FISH)
1. Nuclease-free water.
2. 4% PFA: 4% (w/v) Paraformaldehyde diluted in PBS.
3. 0.5% NP-40: 0.5% (v/v) Nonyl phenoxypolyethoxylethanol
in PBS.
4. SSC buffer: 2Â/1Â/0.2Â Saline sodium citrate, pH 7
(prepared by diluting 20Â saline sodium citrate containing
3 M sodium chloride and 0.3 M sodium citrate in nucleasefree water).
5. Wash buffer: 10% (v/v) Formamide in 2Â SSC buffer.
6. Hybridization buffer: 1% (v/v) Tween 20, 10% (v/v) dextran
sulfate, 50% (v/v) formamide, 500 ng/mL salmon sperm DNA
in 2Â SSC buffer.
7. Telomere leading strand-targeting FISH probes labeled with
carboxytetramethylrhodamine (TAMRA) that are optically distinct from MTSa-targeting MBs (see Note 11). The FISH
probe sequence is TAMRA-CCCTAACCCTAACCCTAA.
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1. HEK293 cells (American Type Culture Collection) (see
Note 8).
2. Cell culture medium: Dulbecco’s modified Eagle’s medium
(DMEM) without phenol red and antibiotics, supplemented
with 10% (v/v) fetal bovine serum (FBS) and 2 mM Lglutamine.
3. Phosphate-buffered saline (PBS): 0.2 g/L KCl, 0.24 g/L
KH 2 PO 4 , 8 g/L NaCl, and 1.44 g/L Na 2 HPO 4 (anhydrous)
in nuclease-free water.
4. Trypsin: Phenol red-free solution of 0.25% (w/v) trypsin
diluted in PBS.
5. 6-Well plates.
2.6 Microporation
(See Note 9)
1. Microporator.
2. PBS.
3. Electroporation buffer (see Note 9).
4. Electrolytic buffer (see Note 9).
5. Electroporation chamber (see Note 9).
6. Electroporation tube (see Note 9).
7. Cell culture medium (as in Subheading 2.5, see Note 10).
8. 8-Well chambered cover glass.
9. 10 μg/mL Fibronectin.
10. Refrigerated microcentrifuge.
11. Cell-counting device.
2.7 Fluorescence In
Situ Hybridization
(FISH)
1. Nuclease-free water.
2. 4% PFA: 4% (w/v) Paraformaldehyde diluted in PBS.
3. 0.5% NP-40: 0.5% (v/v) Nonyl phenoxypolyethoxylethanol
in PBS.
4. SSC buffer: 2Â/1Â/0.2Â Saline sodium citrate, pH 7
(prepared by diluting 20Â saline sodium citrate containing
3 M sodium chloride and 0.3 M sodium citrate in nucleasefree water).
5. Wash buffer: 10% (v/v) Formamide in 2Â SSC buffer.
6. Hybridization buffer: 1% (v/v) Tween 20, 10% (v/v) dextran
sulfate, 50% (v/v) formamide, 500 ng/mL salmon sperm DNA
in 2Â SSC buffer.
7. Telomere leading strand-targeting FISH probes labeled with
carboxytetramethylrhodamine (TAMRA) that are optically distinct from MTSa-targeting MBs (see Note 11). The FISH
probe sequence is TAMRA-CCCTAACCCTAACCCTAA.
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361