HeLa tet-off cell lines (Clontech), in which expression from pTRE vectors is regulated by doxycycline, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; J R Scientific, Woodland, CA), 200 μg/ml G418 (Nacalai Tesque, Kyoto, Japan), 100 units/ml penicillin G (Sigma-Aldrich), and 100 μg/ml streptomycin (Sigma-Aldrich) in a humidified 5% CO2/95% air atmosphere at 37°C. HeLa tet-off cell lines for doxycycline-regulated expression of YFP, SOD1-WT-YFP, or SOD1-G85R-YFP were selected in medium supplemented with 500 μg/ml HygroGold (Invivo-Gen, San Diego, CA) and 1.0 μg/ml doxycycline (Sigma-Aldrich). For transfections, cells were seeded on 3.5-cm dishes (BD, Franklin Lakes, NJ) or 3.5-cm glass-based dishes (Asahi-Technoglass, Tokyo, Japan) 1 day before transfection. All constructs were transfected using Effectene (Qiagen, Dusseldorf, Germany). For photoactivation analysis, mixtures of pTRE-SOD1-G85R-mPAGFP and pTRE-SOD1-G85R-TagRFP constructs (1:1) were transfected into HeLa tet-off cells. For FRET-FLIM assays, pTRE-SOD1-G85R-cp173mVenus or the control pTRE-SOD1-G85R-mVenus was cotransfected with pTRE-SOD1-G85R-mTFP1 at a ratio of 3:1.
Doxycycline
Manufactured by InvivoGen
Sourced in France, Germany
Doxycycline is a broad-spectrum tetracycline antibiotic. It is a highly effective antimicrobial agent used to treat a variety of bacterial infections.
Automatically generated - may contain errors
Lab products found in correlation
Blasticidin, by InvivoGen (3 mentions)
Doxycycline, by Merck Group (2 mentions)
Puromycin, by InvivoGen (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Hygrogold, by InvivoGen (1 mentions)
3.5 cm dishes, by BD (1 mentions)
Penicillin g, by Merck Group (1 mentions)
Effectene, by Qiagen (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Glycid ether 100, by Serva Electrophoresis (1 mentions)
Strep tactin beads, by IBA Lifesciences (1 mentions)
Hygromycin, by InvivoGen (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Normocin, by InvivoGen (1 mentions)
Pierce bca protein assay kit, by Merck Group (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
5 protocols using doxycycline
1
HeLa Cells for Protein Expression
2
Generation of CFBE41o- Cells Expressing Mutant CFTR
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CFBE41o− (cystic fibrosis bronchial epithelial) cells stably expressing DD/AA-CFTR were generated as previously described [18 (link)]. Briefly, CFTR cDNA bearing D565A and D567A (DD/AA) was introduced, cloned into the lentiviral expression vector pLVX-Puro by homologous recombination using the In-Fusion HD Cloning Kit (Clontech-Takara, #121416, Saint-Germain-en-Laye, France). For lentiviral particles’ production, plasmids with the correct sequence were used to calcium phosphate-transfect HEK 293T cell. Lentiviral particles were collected 48 h after transfection and were used to transduce parental CFBE41o− cells [18 (link)].
CFBE41o− cells expressing double-tagged mCherry-FLAG-CFTR (wt, F508del, F508del-4RK or DD/AA) under a doxycycline inducible promoter were grown in DMEM with 4.5 g/L glucose andl -glutamine supplemented with 10% (v/v) FBS, 2 µg/mL puromycin, and 10 µg/mL blasticidin (InvivoGen, #ant-bl, Toulouse, France). CFTR expression was induced with doxycycline 1 µg/mL (Sigma #9891, Gillingham, Dorset, UK) (24-h treatment for wt-CFTR and 48-h treatment for CFTR mutants). All cell lines were grown at 37 °C in 5% CO2. CFTR levels were measured in each cell using the fluorescence quantification to determine total CFTR, given by the mCherry fluorescence intensity and PM CFTR, given by Cy5 fluorescence intensity [19 (link)].
CFBE41o− cells expressing double-tagged mCherry-FLAG-CFTR (wt, F508del, F508del-4RK or DD/AA) under a doxycycline inducible promoter were grown in DMEM with 4.5 g/L glucose and
3
Comprehensive Reagent Inventory for Cell Research
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All reagents used in this study were from Sigma–Aldrich unless otherwise indicated.
Antibiotics: normocin, blasticidin, hygromycin, puromycin, and doxycycline were from Invivogen (San Diego, CA). Restriction endonucleases and specific reagents for cloning, pierce BCA protein assay kit, glutaraldehyde, lead citrate, propylene oxide, and osmium tetroxide were from Merck (Darmstadt, Germany), protein G magnetic beads from NEB (#S1430s), ProtA/G agarose and DMEM (#41966‐029) from Thermo Fisher Scientific (NativeMark™ #LC0725) NativePAGE™ (3–12% Bis‐Tris Protein, #BN1001), Turbofect, Lipofectamine 2000, Ca2+ Green 5N, and MitoTracker™ Green FM (#M7514) from Invitrogen, Streptactin beads from IBA lifesciences. Bradford was from BioRad, proteinase inhibitor from Roche (Basel, Switzerland), C12E8 from TCI Europe, TMRM from molecular probes, glycid ether 100 from Serva (Heidelberg, Germany). Fetal bovine serum (FBS) and pen/strep were from Gibco. Mycoplasma test kit was from MycoAlert Lonza kit, SNARF/AM (C1272; Invitrogen). The working concentration of ruthenium red was calculated with Lambert–Beer law, A = 533 nm, I = 1 cm, Ɛ = 65,000.
Antibiotics: normocin, blasticidin, hygromycin, puromycin, and doxycycline were from Invivogen (San Diego, CA). Restriction endonucleases and specific reagents for cloning, pierce BCA protein assay kit, glutaraldehyde, lead citrate, propylene oxide, and osmium tetroxide were from Merck (Darmstadt, Germany), protein G magnetic beads from NEB (#S1430s), ProtA/G agarose and DMEM (#41966‐029) from Thermo Fisher Scientific (NativeMark™ #LC0725) NativePAGE™ (3–12% Bis‐Tris Protein, #BN1001), Turbofect, Lipofectamine 2000, Ca2+ Green 5N, and MitoTracker™ Green FM (#M7514) from Invitrogen, Streptactin beads from IBA lifesciences. Bradford was from BioRad, proteinase inhibitor from Roche (Basel, Switzerland), C12E8 from TCI Europe, TMRM from molecular probes, glycid ether 100 from Serva (Heidelberg, Germany). Fetal bovine serum (FBS) and pen/strep were from Gibco. Mycoplasma test kit was from MycoAlert Lonza kit, SNARF/AM (C1272; Invitrogen). The working concentration of ruthenium red was calculated with Lambert–Beer law, A = 533 nm, I = 1 cm, Ɛ = 65,000.
4
Mitochondrial Dynamics Regulation Assay
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Chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany) except Mdivi-1 (Enzo Life Science, Lörrach, Germany) as well as doxycycline, blasticidin and zeocin (Invivogen, San Diego, CA, USA). Cell culture materials and media were obtained from Biochrom (Berlin, Germany). MitoTracker GreenFM (MTG) and MitoSOX™ Red superoxide indicator (MitoSOX) were purchased from Molecular Probes (Eugene, USA).
The following primary antibodies were used: rabbit anti-DNP (Sigma-Aldrich, Deisenhofen, Germany), rabbit anti-PINK1 (Cell Signaling, Boston, MA, USA), mouse anti-β-actin (Cell Signaling, Boston, USA), rabbit anti-COX IV (Cell Signaling, Boston, MA, USA), rabbit anti-Lon protease (Abcam, Cambridge, UK), mouse anti-GAPDH (Abcam, Cambridge, UK), rabbit anti-Ki-67 (Abcam, Cambridge, UK), mouse anti-CDKN2A/p16INK4α (Abcam, Cambridge, UK), rabbit anti-p21 Waf1/Cip1 (Cell Signaling, Boston, MA, USA), mouse anti-MT-CO1 (Abcam, Cambridge, UK), mouse anti-SDHA (Abcam, Cambridge, UK), mouse anti-VDAC (Abcam, Cambridge, UK) and rabbit anti-Fis1 (Cell Signaling, Boston, MA, USA). Secondary antibodies used for immunoblotting were purchased from LI-COR Biosciences (Lincoln, AL, USA). The FITC-labeled antibody for immunofluorescence was purchased from Invitrogen (Carlsbad, CA, USA).
The following primary antibodies were used: rabbit anti-DNP (Sigma-Aldrich, Deisenhofen, Germany), rabbit anti-PINK1 (Cell Signaling, Boston, MA, USA), mouse anti-β-actin (Cell Signaling, Boston, USA), rabbit anti-COX IV (Cell Signaling, Boston, MA, USA), rabbit anti-Lon protease (Abcam, Cambridge, UK), mouse anti-GAPDH (Abcam, Cambridge, UK), rabbit anti-Ki-67 (Abcam, Cambridge, UK), mouse anti-CDKN2A/p16INK4α (Abcam, Cambridge, UK), rabbit anti-p21 Waf1/Cip1 (Cell Signaling, Boston, MA, USA), mouse anti-MT-CO1 (Abcam, Cambridge, UK), mouse anti-SDHA (Abcam, Cambridge, UK), mouse anti-VDAC (Abcam, Cambridge, UK) and rabbit anti-Fis1 (Cell Signaling, Boston, MA, USA). Secondary antibodies used for immunoblotting were purchased from LI-COR Biosciences (Lincoln, AL, USA). The FITC-labeled antibody for immunofluorescence was purchased from Invitrogen (Carlsbad, CA, USA).
5
Generation of Recombinant SARS-CoV-2 Variants
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Recombinant SARS-CoV-2 was generated by CPER as previously described.20 (link) In brief, the 9 fragments of SARS-CoV-2 and UTR linker for SARS-CoV-2 described above were prepared by PCR using PrimeSTAR GXL DNA polymerase (Takara Bio). To prepare recombinant SARS-CoV-2 expressing the genes of MA10, B.1.351.1, BA.1, XBB.1.5, NanoLuc, or Akaluc mutations, the respective F3, F4, F8, or F9-10 plasmids were used for CPER. To generate recombinant SARS-CoV-2, the CPER products (25 μL) were transfected into HEK293-3P6C33 cells with Opti-MEM (Thermo Fisher Scientific) and TransIT-LT1 (Mirus Bio) according to the manufacturer’s protocol. At 6 h post-transfection, the culture medium was replaced with DMEM containing 2% FBS, 1% PS, and doxycycline (1 μg/mL) (InvivoGen). All viruses were stored at −80°C until use.
The infectious titers in culture supernatants were determined by quantifying the 50% tissue culture infectious dose (TCID50).49 Cell culture supernatants were used to inoculate naive VeroE6/TMPRSS2 cells in 96-well plates after 10-fold serial dilution with DMEM containing 1 mg/mL G418 (Nacalai Tesque) and 2% FBS, and the infectious titers were determined at 72 h post-infection (hpi).
The infectious titers in culture supernatants were determined by quantifying the 50% tissue culture infectious dose (TCID50).49 Cell culture supernatants were used to inoculate naive VeroE6/TMPRSS2 cells in 96-well plates after 10-fold serial dilution with DMEM containing 1 mg/mL G418 (Nacalai Tesque) and 2% FBS, and the infectious titers were determined at 72 h post-infection (hpi).
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