Selection of Red Fluorescent Protein for Genetic Labeling of Mitochondria and Intercellular Transfer of Viable Mitochondria – Scientific Reports

cell culture

HEK293T cells were purchased from GeneHunter and cultured on collagen-coated dishes (Nunc) in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and streptomycin and 2.5 ng/ml amphotericin B (Gibco). . .

Previously established immortalized hAEC lineage cells were used30. Briefly, primary human amniotic epithelial cells from five different donors were immortalized using an SV40 lentiviral vector (pLenti-SV40-T+t, Applied Biological Materials Inc., Richmond, BC, Canada). The established immortalized hAEC lines were evaluated morphologically and one line (iAE129) was selected and used for further studies.

iAECs were cultured in DMEM supplemented with 10% fetal bovine serum (Nichirei), 200 µM I-Glutamine (Gibco), 1x Non-Essential Amino Acids (Gibco), 5.5 µM 2-mercaptoethanol (Gibco), 5 ng/mL EGF (PeproTech), 100 U/mL penicillin and streptomycin, and 2.5 ng/mL amphotericin B.

All cells were cultured in 5% CO22 at 37 degrees.

Lentiviral vector production

Lentiviral vectors (transfer plasmids) were constructed by cloning each fluorescent gene into a backbone of a second generation common lentiviral vector plasmid with hPGK promoter (pLKO). Two human COXA8 genes were inserted as tandem MTS under the promoter (Fig. 1).

For lentiviral particle production, HEK293T cells were seeded onto collagen-coated 10 cm dishes at 70-80% confluency. Cells were co-transfected with 4 µg lentiviral vector plasmid along with 4 µg psPAX2 and 2 µg pMD2. G (Packaging plasmids, Addgene plasmids 12.260 and 12.259). Transfection mixes were prepared in 500 µl Opti-MEM I (Life Technologies) containing the DNA and 27 µl Lipofectamine 3000 (Life Technologies) according to the manufacturer’s protocol. The medium was changed 12 h after transfection and lentiviral supernatants were collected at 24 and 48 h. Collected lentiviral supernatants were concentrated by ultracentrifugation and the medium was exchanged with phosphate buffered saline.

imaging

To image mitochondrial-targeted proteins, iAECs were seeded in a 24-well plate (Nunc) at a density of 50,000 cells/well and infected with lentivirus after 12 h. Cells were seeded on glass bottom dishes (Matsunami) 2 days prior to imaging. The cells were loaded with 100 nM MitoTracker Green or Orange CMTMRos (Life Technologies) and 500 ng/ml Hoechst 33342 for 30 min in culture medium at 37°C. For lysosome staining, cells were loaded with 100 nM LysoTracker Deep Red (Life Technologies) in addition to the above. Then the cells were washed twice with medium and imaged in 5% CO 22 at 37°C in culture medium.

For the mitochondrial transfer experiment, HEK293T cells transduced with cytosolic EGFP were transferred to collagen and poly-I-Lysine coated glass bottom dishes for two days. Cells were treated with 300 μM hydrogen peroxide for 60 min at 37 °C and then co-cultured with iAECs transduced with mitochondrial-targeted TurboRFP in fresh iAECs medium. After 20 h of culture, cells were loaded with MitoTracker Deep Red (Life Technologies) and imaged at room temperature. We performed mitochondrial transfer experiments three times and found mitochondrial transfer in all experiments.

All fluorescence images were acquired with a confocal microscope (TCS SP8, Leica) equipped with a 63× objective (NA 1.40, HC PL APO, Leica) with excitation/emission wavelengths (nm) of 405/415–455 for Hoechst 33342 , 488/496–543 for MitoTracker Green and EGFP, 555/565–620 for MitoTracker Red and the red fluorescent proteins, and 638/570–700 for LysoTracker Deep Red and MitoTracker Deep Red.

3D reconstructed images were obtained with Leica LAS X software (version 3.5.5) and other images were analyzed with imageJ software (version 1.52p).

Cell growth and flow cytometry experiments

iAECs were seeded in a 24-well plate at a density of 50,000 cells/well and infected with lentiviruses at an MOI of 5. After 4 days of infection, the cells were passaged and cultured for 6 days to rule out viral toxicity. Then the cells were seeded again in 96-well plates (Nunc) at a density of 5000 cells/well. Cell proliferation was measured at 12, 24, 48, 72 and 120 h after seeding using a Cell Counting Kit-8 (Dojindo) according to the manufacturer’s protocol.

For flow cytometric analysis, cells were analyzed using a FACS Aria (BD) equipped with 488 (for EGFP) or 561 (for DsRed, mCherry, TurboRFP and mKOκ) nm laser and 530/30 (for EGFP) or 582/15 (for DsRed, mCherry, TurboRFP and mKOκ) nm emission filters. The cells were analyzed 6, 14, 21, 28 and 42 days after infection. Cells were passaged at 1 week intervals.

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