Doxycycline

Manufactured by Cayman Chemical

Sourced in United States

Doxycycline is a tetracycline antibiotic used for laboratory research purposes. It functions as a protein synthesis inhibitor.

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Lab products found in correlation

Tr 1003 g, by Merck Group (3 mentions) Hygromycin, by Cayman Chemical (2 mentions) Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions) Anhydrochlortetracycline hcl, by Cayman Chemical (2 mentions) Continuum transfection reagent, by Gemini Bio (2 mentions) Fetal bovine serum (fbs), by Gemini Bio (2 mentions) Rpmi 1640 media, by American Type Culture Collection (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Neomycin, by Thermo Fisher Scientific (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Doxycycline, by Thermo Fisher Scientific (1 mentions) Hygromycin, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Lonza (1 mentions) Penicillin streptomycin, by Biowest (1 mentions) Polybrene, by Merck Group (1 mentions) Puromycin, by Merck Group (1 mentions) Lv003, by Applied Biological Materials (1 mentions) Lentifectin, by Applied Biological Materials (1 mentions) Nifedipine, by Cayman Chemical (1 mentions) Metformin, by Targetmol (1 mentions)

15 protocols using doxycycline

Synchronization and Perturbation of Cell Lines

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HEK293T, U2OS and CSM14.1 cells were grown in DMEM (Lonza) supplemented with 10% FBS and 1% penicillin-streptomycin (Biowest). CMS14.1 cells were additionally grown in 0.1 mg/ml neomycin, 0.1 mg/ml hygromycin (Alfa Aesar), 4 μg/ml puromycin (Alfa Aesar) and 1 μg/ml doxycycline to maintain selection the CSM14.1 clonal cell lines. SCA3-Q23 or SCA3-Q70 expression was induced by the withdrawal of doxycycline (Cayman Chemical). ΔATXN3 U2OS cells were described before (16 (link)).
G1/S boundary synchronization was performed by treating cells with 2.5 mM thymidine (Merk-Millipore) for 24 h. Cells were treated with 60 μM CDC7i (PHA767491, Cayman Chemical Company) for 4 h, 80 mM sucrose (Merk-Millipore) for 30 min or 1 h, HDAC3 inhibitor RGFP966 2 μM (HY-13909, MedChemExpress) for 24 h and 20 μg/ml cycloheximide (CHX, Merck Millipore) for the indicated times.

Hernández‐Carralero E., Cabrera E., Rodríguez-Torres G., Hernández-Reyes Y., Singh A.N., Santa-María C., Fernández-Justel J.M., Janssens R.C., Marteijn J.A., Evert B.O., Mailand N., Gómez M., Ramadan K., Smits V.A, & Freire R. (2023). ATXN3 controls DNA replication and transcription by regulating chromatin structure. Nucleic Acids Research, 51(11), 5396-5413.

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Generation and Maintenance of Nrf2-knockdown Cells

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The lentiviral vector of Nrf2-shRNA (shNrf2) was packaged into virus particles by the method reported previously^{37 (link)}, which was provided by Sang-Min Jeon, Professor, College of Pharmacy, Ajou University (Gyeonggido, Rep. of Korea). 293T cells were transfected with a lentiviral vector using Lipofectamine® 2000 according to the recommended protocol on the Addgene website. Lentivirus-containing conditioned medium (LCCM) was aliquoted into 1 ml stock in each cryovial. Then, HeLa stable cells that do not express Nrf2 (tet-shNrf2, + Dox) were prepared as follows. Briefly, HeLa 1 × 10⁵ cells were incubated in each well of the 6-well plate overnight. Cell culture in 500 μl medium of each well was mixed with 1 ml LCCM and 1.2 μl polybrene (Millipore TR-1003-G). Culture medium was changed with 2 ml fresh medium containing 250 μg/ml hygromycin (Cayman 14,291). The infected shNrf2-positive control cells (tet-shNrf2, -DOX) were selected by the treatment with hygromycin every 3 days. Nrf2-knockdown (KD) cells were obtained and maintained by the treatment with 0.2 μg/ml doxycycline (Cayman 14,422) every 2 days.

Lee J.W., Thuy P.X., Koo J.H, & Moon E.Y. (2022). Nuclear factor (erythroid-derived 2)-like 2 counter-regulates thymosin beta-4 expression and primary cilium formation for HeLa cervical cancer cell survival. Scientific Reports, 12, 20170.

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Lentiviral Transduction of F-Tractin for Actin Visualization

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To visualize actin polymers in live cells after encapsulation in gel coatings while minimally impacting baseline cellular functions, the plasmid containing F‐tractin fused with the fluorescent gene tdTomato (a gift from Andrei Karginov, UIC) was cloned into the lentiviral vector pCW57.1 (a gift from David Root, Addgene plasmid #41393) for inducible expression in the presence of doxycycline. F‐tractin is known to selectively bind to polymerized F‐actin.^[⁵⁸^] Lentiviral particles were produced with a second‐generation lentiviral packaging system (LV003, Applied Biological Materials) using a transfection reagent (Lentifectin, Applied Biological Materials) in HEK293T cells. Lentiviral particles were purified and applied to D1 MSCs at passage 5 with polybrene (8 µg mL⁻¹; Sigma) for 3 days, followed by the selection of transduced cells by puromycin (5 µg mL⁻¹; Sigma) for 7 days and sorting of tdTomato⁺ cells after doxycycline (500 ng mL⁻¹; Cayman Chemical) treatment for 1 day.

Cho I.S., Gupta P., Mostafazadeh N., Wong S.W., Saichellappa S., Lenzini S., Peng Z, & Shin J. (2022). Deterministic Single Cell Encapsulation in Asymmetric Microenvironments to Direct Cell Polarity. Advanced Science, 10(3), 2206014.

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Inducible Fibroblast Lines for CRISPR/Cas9 Studies

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MKP1- or GFP-overexpressing fibroblast lines were generated by infection with lentiviral particles containing pLVX-TetOne-Puro-hDUSP1 or pLVX-TetOne-Puro-eGFP (Addgene) plasmids. Stable inducible fibroblast lines capable of CRISPR/Cas9-mediated deletion of MKP1, p38α, and p38γ were generated via infection with lentiviral particles containing pLentiCRISPR v2 TLCV2 plasmids (Addgene no. 87360) controlling the following sgRNA sequences: DUSP1, 5′-CGTCCAGCAACACCACGGCG-3′; MAPK14, 5′-CACAAAAACGGGGTTACGTG-3′; and MAPK12, 5′-CTCATGAAACATGAGAAGCT-3′. The NT sequence 5′-AAATGTGAGATCAGAGTAAT-3′ (Thermo Fisher Scientific, A35526) was likewise incorporated into TLCV2 and used as a negative control. Polybrene (2 μg/mL, MilliporeSigma, TR-1003-G) or protamine sulfate (8 μg/mL, MilliporeSigma, P4020) was added to lentiviral suspensions to enhance transduction efficiency in MRC5 (normal fetal HLFs) and primary IPF cells, respectively, followed by puromycin selection (0.8 μg/mL) for 48–72 hours. Conditional overexpression or CRISPR/Cas9-mediated deletion of the aforementioned genes was achieved by addition of doxycycline (1 μg/mL, Cayman Chemicals) to conditioned medium.

Fortier S.M., Walker N.M., Penke L.R., Baas J.D., Shen Q., Speth J.M., Huang S.K., Zemans R.L., Bennett A.M, & Peters-Golden M. (2024). MAPK phosphatase 1 inhibition of p38α within lung myofibroblasts is essential for spontaneous fibrosis resolution. The Journal of Clinical Investigation, 134(10), e172826.

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Zebrafish Pharmacological Compound Screening

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Concentrated stock solutions for pentylenetetrazole (PTZ, Sigma), metformin (Cayman Chemical), doxycycline (Cayman Chemical), nifedipine (Cayman Chemical), and pyrantel tartrate (TargetMol) were made by dissolving each in either ddH2O (PTZ, doxycycline, metformin) or DMSO (nifedipine, pyrantel tartrate). 7 dpf (or 5 dpf, in the case of the doxycycline treatment experiments) zebrafish larvae were placed individually into separate wells of a 96‐well plate in fish water. All water in each well was carefully drawn off, and 250 μL 1X Ringer's Solution (116 mmol/L NaCl, 2.9 mmol/L KCl, 1.8 mmol/L CaCl2, and 5 mmol/L HEPES) was pipetted into the well. Lastly, small volumes of concentrated stock solutions of drug (or vehicle) were delivered to their respective groups to reach their final concentrations: 5 mmol/L PTZ, 2 μmol/L metformin, 10 μmol/L doxycycline, 12 μmol/L nifedipine, and 75 μmol/L pyrantel tartrate. Solutions were mixed by gently tapping the plate. The larvae were divided such that all five groups had approximately equal representation on every plate (from 10 to 20 embryos per treatment group per plate).

Brueggeman L., Sturgeon M.L., Martin R.M., Grossbach A.J., Nagahama Y., Zhang A., Howard MA I.I.I., Kawasaki H., Wu S., Cornell R.A., Michaelson J.J, & Bassuk A.G. (2018). Drug repositioning in epilepsy reveals novel antiseizure candidates. Annals of Clinical and Translational Neurology, 6(2), 295-309.

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Targeted Protein Degradation Pathway

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The cherry-picked library including the inhibitors trametinib, RO5126766, and ulixertinib were obtained from Selleckchem (Houston, TX, USA). The degrader dTAG13 was purchased from Tocris/Bio-Techne GmbH (Wiesbaden, Germany) and Doxycycline from Cayman Chemical (Ann Arbor, MI, USA).

Schneeweis C., Diersch S., Hassan Z., Krauß L., Schneider C., Lucarelli D., Falcomatà C., Steiger K., Öllinger R., Krämer O.H., Arlt A., Grade M., Schmidt-Supprian M., Hessmann E., Wirth M., Rad R., Reichert M., Saur D, & Schneider G. (2022). AP1/Fra1 confers resistance to MAPK cascade inhibition in pancreatic cancer. Cellular and Molecular Life Sciences, 80(1), 12.

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Tat Transgene Induction in Mice

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To induce expression of the tat transgene (or not), Tat(−) and Tat(+) mice were administered doxycycline hylcate (prepared fresh daily and dissolved to 30 mg/kg, i.p., in 0.9% sterile saline; Cayman Chemical, Ann Arbor, MI) for 5 days, followed by 2 days without manipulation for doxycycline washout. Starting on day 8, the estrous cycle was assessed daily.

Salahuddin M.F., Qrareya A.N., Mahdi F., Jackson D., Foster M., Vujanovic T., Box J.G, & Paris J.J. (2019). Combined HIV-1 Tat and Oxycodone Activate the Hypothalamic-Pituitary-Adrenal and -Gonadal Axes and Promote Psychomotor, Affective, and Cognitive Dysfunction in Female Mice. Hormones and behavior, 119, 104649.

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Prostate Cancer Cell Line Maintenance and SBP1 Expression

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The PC-3 human prostate carcinoma cell line was maintained in RPMI-1640 media (Gibco), and LNCaP human prostate carcinoma cell line was maintained in RPMI-1640 media (American Type Culture Collection). All media were supplemented with 10% fetal bovine serum (Gemini Bio), 100 U/mL penicillin, and 100ug/mL streptomycin, and cells were maintained at 37°C with 5% CO₂. Cell lines were authenticated by Genetica DNA Laboratories (Burlington, NC). The constitutively-active and inducible SBP1 expression constructs were introduced via transfection using Continuum™ Transfection Reagent (Gemini Bio) into PC-3 cells, and PC-3 cells that were previously infected with the tetracycline trans-activator (TETON) construct (11 (link)), respectively. The same reagent was also used for the transfection of plasmids into LNCaP cells. Transfected cells were selected in 500ug/mL G418 (Sigma-Aldrich), and expanded and screened for SBP1 expression by western blotting and qRT-PCR using SBP1 forward primer (5’-CCAAAGCTGCACAAGGTCAT-3’), SBP1 reverse primer (5’- CATCCAGCAGCACAAAACCC-3’), RPLP0 forward primer (5’- CCTCGTGGAAGTGACATCGT-3’), and RPLP0 reverse primer (5’- CTGTCTTCCCTGGGCATCAC-3’). Ectopic expression of SBP1 was induced following incubation with 0.5ug/mL doxycycline or 0.05ug/mL anhydrochlortetracycline-HCl (Cayman Chemical) for 48–72 hours.

Elhodaky M., Hong L.K., Kadkol S, & Diamond A.M. (2020). Selenium‐binding protein 1 alters energy metabolism in prostate cancer cells. The Prostate, 80(12), 962-976.

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Cellular Responses to Oxidative Stress

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Hygromycin B and doxycycline were purchased from Cayman (Ann Arbor, MI, USA). N-acetyl-l-cysteine (NAC) and antibodies to β-tubulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). HIF- inhibitor (sc-205346) and antibodies to HIF-1α (sc-10790) or Nrf2 (sc-365949) were obtained from Santa Cruz Biotechnology (CA, USA). Antibodies to CXCR4 (35-8800) were obtained from Invitrogen, Thermo Fisher Scientific. (Waltham, MA, USA). 2′,7′–dichlorofluorescin diacetate (DCF-DA) was purchased from Molecular Probe (Eugene, OR, USA). CellTiter-Glo substrates and ViaFect™ were purchased from Promega Co. (Madison, WI, USA). pCDNA3.1-Nrf2 plasmids were kindly provided from Professor Byung-Chul Kim, Division of Life Sciences, Kangwon National University, Chuncheon, Republic of Korea. Except indicated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA)^{41 (link)}.

Jang J.W., Thuy P.X., Lee J.W, & Moon E.Y. (2021). CXCR4 promotes B cell viability by the cooperation of nuclear factor (erythroid-derived 2)-like 2 and hypoxia-inducible factor-1α under hypoxic conditions. Cell Death & Disease, 12(4), 330.

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MRSA Antibiotic Susceptibility Assay

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MRSA cultures were prepared by inoculating single colonies into 2 mL of LB broth and grown for 16 h at 37°C with constant shaking at 250 rpm. The overnight cultures were diluted 1:20 into fresh LB broth, grown until they reached the exponential phase (OD_600nm: 0.4–0.7), and diluted 1:10 into a sterile 96-well plate (Fisher Scientific) containing a range of antibiotic concentrations in LB broth. The antibiotics that were used were: vancomycin (VWR, Suwanee, GA, USA), doxycycline (Cayman Chemicals, Ann Arbor, MI, USA), daptomycin (Fisher Scientific), and methicillin (Fisher Scientific). Plates were incubated at 37.0°C for 24 h in a BioTek synergy microplate reader, which was set to constantly shake at medium intensity and absorbance was recorded at 630 nm every 30 min.

Chittams-Miles A.E., Malik A., Purcell E.B, & Muratori C. (2023). Nanosecond pulsed electric fields increase antibiotic susceptibility in methicillin-resistant Staphylococcus aureus. Microbiology Spectrum, 12(1), e02992-23.

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