The pDsRed2-Mito was obtained from Clontech (Mountain View, CA, USA) and the pcDNA3-DRP1K38A plasmid in baculovirus expression vector (US National Center for Biotechnology Information accession number NM_005690.3) was provided by Dr AM van der Bliek. For transfection of primary RGCs, 100 μl of Nucleofector Solution (Lonza, Allendale, NJ, USA) was mixed with 1 × 106 cells and then 1 μg of pcDNA3 and pcDNA3-DRP1 were transfected using a Nucleofector II/2b Device (Lonza).
Pdsred2 mito
Manufactured by Takara Bio
Sourced in United States
PDsRed2-Mito is a red fluorescent protein that localizes to the mitochondria. It can be used to visualize mitochondrial structure and dynamics in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Pegfp c1, by Takara Bio (4 mentions)
Pdsred2 er, by Takara Bio (3 mentions)
Pcaggs n ha vector, by Addgene (2 mentions)
Pdsred2 golgi, by Takara Bio (2 mentions)
Pdsred2 lamp1, by Takara Bio (2 mentions)
Pdsred2 peroxi, by Takara Bio (2 mentions)
Flag tag (flag), by Beyotime (2 mentions)
Pcmv vector, by Beyotime (2 mentions)
Prl tk, by Promega (2 mentions)
Pspax2, by Addgene (2 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Pcdh cmv mcs ef1 puro, by System Biosciences (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Nucleofector 2 2b device, by Lonza (1 mentions)
Nucleofector solution, by Lonza (1 mentions)
Mcherry parkin, by Addgene (1 mentions)
Peasy basic seamless cloning and assembly kit, by Transgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
27 protocols using pdsred2 mito
1
Transfection of Primary Retinal Ganglion Cells
2
ASFV Strain China/2018/AnhuiXCGQ Constructs
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All the ORFs of the ASFV strain China/2018/AnhuiXCGQ (GenBank accession number MK128995.1 ), including 11 members belonging to the MGF110, were generated by gene synthesis (GENEWIZ, Suzhou, China) and cloned into the pCMV vector with a C-terminal FLAG tag (Beyotime Biotechnology, Shanghai, China). The ATF4-RLuc and ATF4-EGFP reporters were constructed as previously described (31 (link)). Briefly, a full-length human ATF4 mRNA 5′ leader and ATF4 initiation codon were subcloned into pRL-TK (Promega, WI) and pEGFP-C1 (Clontech, Mountain View, CA), respectively. cDNAs of PDIA3, TMED4, and PSMA4 were amplified from total mRNA of PK-15 cells by conventional RT-PCR and inserted into the KpnI and XhoI sites of the pCAGGS-N-HA vector (Addgene, MA) to obtain hemagglutinin (HA)-tagged constructs. All constructed plasmids were verified by sequencing.
The organelle markers, including pDsRed2-ER, pDsRed2-Golgi, pDsRed2-Mito, pDsRed2-LAMP1, and pDsRed2-Peroxi vectors that encode targeting sequences of the corresponding organelles, were purchased from Clontech.
The organelle markers, including pDsRed2-ER, pDsRed2-Golgi, pDsRed2-Mito, pDsRed2-LAMP1, and pDsRed2-Peroxi vectors that encode targeting sequences of the corresponding organelles, were purchased from Clontech.
3
Generation of Lentiviral Vectors and Stable Cell Lines
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The plasmids mCherry-Parkin (Plasmid #23956), GFP-LC3 (Plasmid #11546), psPAX2 (Plasmid #12260) and pMD2.G (Plasmid #12259) were obtained from Addgene (Cambridge, MA, USA). The plasmid pDsRed2-Mito was purchased from Clontech (Mountain View, CA, USA). The cDNA cloning and expression plasmid pCDH-CMV-MCSEF1-Puro was obtained from System Biosciences (Palo Alto, CA, USA). For lentiviral vectors construction, gene fragments were PCR amplified using I-5™ Hi-Fi DNA Polymerase (#PDP-100, MCLAB, San Francisco, CA, USA) and then inserted into pCDH-CMV-MCSEF1-Puro lentivirus vector using pEASY®-Basic Seamless Cloning and Assembly Kit (#CU201-02, TransGen Biotech, Beijing, China). Then, the sequenced lentivirus plasmid was co-transfected with packaging plasmids psPAX2 and pMD2.G into HEK293T/17 using NEOFECT™ DNA transfection reagent (Neofect biotech, Beijing, China) for 48–72 h. The culture supernatants were harvested and inoculated into EA.hy926 cells. The stable cell lines were generated by two rounds of puromycin selection.
4
Visualizing Mitochondrial Dynamics in HeLa Cells
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For immunostaining, HeLa cells were transfected with pDsRed2-Mito (Clontech) using lipofectamine 2000 (Invitrogen) and grown on glass coverslips for 2 days before fixation in 4% paraformaldehyde, blocking in Abdil (PBS, 0.2% Triton X-100, 3% BSA (Sigma)) and immunolabeling with anti-C7orf55 (Abcam ab188310). Secondary donkey anti-rabbit IgG A488-conjugated antibodies (Abcam ab150073) were used and cells were labeled with Hoescht 33342 (Sigma). Microscopy was performed using a Zeiss LSM700 confocal microscope.
5
Generating Mitochondrial-Deficient PC12 Cells
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PC12 cells (rat pheochromocytoma cells, clone 251) were cultured as described.6 (link) To obtain cells lacking mtDNA (ρ0 cells), PC12 cells with a low passage number were cultured for 1 month in medium containing 0.1 μg/ml ethidium bromide and 50 μg/ml uridine (Sigma-Aldrich Corp., St. Louis, MO, USA).36 (link) During this period, cells were passaged once a week. PC12 cells were transiently transfected with the expression vector pDsRed2-mito (Clontech Laboratories, Palo Alto, CA, USA) by using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) or pEB3-mCherry (EUROSCARF, Frankfurt, Germany) by using electroporation.
6
Establishing Stable Fluorescent Mitochondrial Cell Line
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To generate a stable cell line expressing fluorescently labeled mitochondria, PC12 cells were transfected with pDsRed2-mito (Clontech Laboratories, Inc., 632421) using transfection reagent Fugene HD (Promega Corporation, E2311), according to manufacturer’s protocol. For stable PRKN overexpression, cells were transfected with pcDNA3.1(+)-6MYC-human PRKN. An empty vector, pcDNA3.1(+), was used as control. For stable PRKN knockdown, cells were transfected with Prkn shRNA plasmid (Santa Cruz Biotechnology, sc-270243-SH) [37 (link)]. Control shRNA plasmid (Santa Cruz Biotechnology, sc-108060) was used as control. Forty-eight hours after transfection, cells were cultured with selection-medium containing 500 µg/ml G418 (Wako Pure Chemical Industries, Ltd., 071–06431) or 1 µg/ml puromycin (Sigma-Aldrich, P8833). Selection process was carried out for a series of passaging by introducing fresh medium and antibiotic to the cells. Following 10–14 days, single colonies of stably transfected cells formed were picked up and expanded in culture medium containing 250 µg/ml G418 or 0.5 µg/ml puromycin. DsRed2-labeled mitochondria were confirmed by immunofluorescence microscopy. PRKN overexpression was determined by western blot analysis. To monitor Prkn gene expression knockdown, quantitative polymerase chain reaction (qPCR) and western blot analysis were performed.
7
Immunofluorescence Imaging of Cellular Organelles
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Immunofluorescence of HeLa and mIMCD3 cells was described previously [58 (link)]. Briefly, cells were cultivated in 6 wells (Greiner Bio-One) on cover glasses (Carl Roth) until about 80% confluence. Primary antibody dilutions in PBS: anti-acetyl-α-tubulin 1:100; anti-Pyruvate dehydrogenase 1:1,000; anti-TRPP2 (G-20) 1:1,000; anti-V5 1:1,000. Mitochondria were visualized with pDsRed2-Mito (Clontech) according to the instructions of the manufacturer. A Leica TCS SP8 STED 3X microscope was used for confocal image acquisition. Vertical projections of recorded stacks were generated using ImageJ 2. STED was used to image close contacts between the ER and mitochondria. STED data were rendered in Imaris 8 (Bitplane). Brightfield images were recorded using an Axio Observer microscope (Zeiss).
8
Cloning and Transfection of hMT1A and EGFP Plasmids
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The synthetic HindIII-CAMP-hMT1A-6xHis-BamHI gene (Bioneer, Daejeon, Korea) was cloned into the pcDNA3.1 vector (Promega, Madison, WI, USA) for mammalian expression (pCAMP-hMT1A). The sequence of the synthetic gene was AAG CTT ATG GGC TAT GGC AGG AAG AAG CGG AGA CAG CGA CGA CGA TTG TTG CGC GCT GCC CTG CGC AAG GCT GCC CTG ATG GAC CCC AAC TGC TCC TGC GCC ACT GGT GGC TCC TGC ACC TGC ACT GGC TCC TGC AAA TGC AAA GAG TGC AAA TGC ACC TCC TGC AAG AAG AGC TGC TGC TCC TGC TGC CCC ATG AGC TGT GCC AAG TGT GCC CAG GGC TGC ATC TGC AAA GGG GCA TCA GAG AAG TGC AGC TGC TGT GCC CAT CAT CAT CAT CAT CAT TAG GGA TCC . The PCR fragment of NheI-CAMP-HindIII was cloned into the pcDNA3.1-GFP-N3 vector (Clontech, Mountain View, CA, USA) to prepare the pCAMP-EGFP plasmid. The pCAMP-hMT1A and pCAMP-EGFP plasmids were separately transfected into 70% confluent SK-Hep1 cells in six-well plates using Superfect® transfection reagent (QIAGEN, Valencia, CA, USA). pcDNA3.1-transfected cells were used as a control. SK-Hep1 cells expressing DsRed2-mito were prepared by stable transfection of pDsRed2-mito (Clontech) using the same method28 (link). Stably transfected cells were established by selection using G418 (1000 μg/ml) for 2 weeks.
9
Mitochondrial Dynamics Analysis by Confocal Microscopy
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To label mitochondria, cells were transfected with pDsRed2-Mito (Clontech Laboratories) according to the manufacturer’s instructions using Lipofectamine 2000 (Invitrogen). After 7 h of incubation, the medium containing Lipofectamine was replaced by complete culture medium supplemented with 10% FBS. To examine the role of p32 in mitochondrial dynamics, cells were transfected with p32 siRNA. Mitochondrial morphology was examined under a FV3000 confocal microscope (Olympus), and mitochondrial length was measured using ImageJ software. For each group, approximately 270 mitochondria from 5 to 8 different random fields were measured, and the average length of mitochondria was calculated.
10
Mitochondrial Visualization in Cell Lines
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HepG2 and H9c2 were purchased from American Type Culture Collection (ATCC). Human mesenchymal stem cell (hMSCs) was purchased from Gibco (Carlsbad, CA, USA). HepG2 and R-HepG2 were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics mixture (PS), except that R-HepG2 cells were supplemented with 1.2 μM of DOX in culture medium. H9c2 and hMSCs were cultured in DMEM and alpha-MEM supplemented with 10% FBS and 1% PS, respectively. hMSCs were assayed within four passages. To generate stable cell lines constitutively expressing the mitochondrial-RFP tag for mitochondria visualization, a construct pDsRed2-Mito (Clontech, CA, USA) carrying a human cytochrome C oxidase subunit VIII mitochondrial targeting sequence was transfected to cells using lipofectamine-3000 reagent according to manufacturer’s protocol. After 48 h of incubation, untransfected cells were eliminated by 1 mg/mL of Geneticin (InvivoGen, San Diego, CA, USA). To ensure clonal purity, transfected cells were sorted to a single clone by a cell sorter (FACSMelody, BD Biosciences, San Jose, CA, USA). Cells carrying the mitochondrial-RFP tag were expanded from a single clone. Fluorescence of the RFP-labelled mitochondria was verified by flow cytometry (FACSVerse, BD Biosciences, San Jose, CA, USA) and confocal microscopy (SP8, Leica, Wetzlar, Germany).
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