Hygromycin

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, France, Italy, Belgium, Spain

Hygromycin is a pharmaceutical compound used as a selective antibiotic for bacterial and eukaryotic cell cultures. It functions by inhibiting protein synthesis, which is essential for cell growth and survival. This compound is commonly used in research applications to maintain selection pressure and ensure the stability of genetically modified cell lines.

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Lab products found in correlation

503 protocols using hygromycin

Stable Transfection and Selection of hRAD21 Variants

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R32-hRAD21
HEK293T cells were seeded at a density of 4x10⁵ cells and stably transfected with 4 µg of Vector [48 (link)] (R32-hRAD21 or R32-hRAD21 p.P298S or R32-hRAD21 p.P298A using Lipofectamine2000 (Invitrogen) and selected with Hygromycin (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 200 µg/mL for 7 days. Continuous culturing was performed with Hygromycin concentration altering between 100 µg/mL and 200 µg/mL, put freshly 3 times a week.
pRTS-1-RAD21
HEK293T cells were seeded at a density of 5x10⁵ cells and stably transfected with 4 µg of Vector [27 (link)] (pRTS-1-RAD21, pRTS-1-RAD21 p.P298S or pRTS-1-RAD21 p.P298A using Lipofectamine2000 (Invitrogen) and selected with Hygromycin (Invitrogen) at a concentration of 400 µg/mL for 7 days. Continuous culturing was performed with Hygromycin at concentrations altering between 200 µg/mL and 400 µg/mL, put freshly 3 times a week.

Schedel A., Friedrich U.A., Morcos M.N., Wagener R., Mehtonen J., Watrin T., Saitta C., Brozou T., Michler P., Walter C., Försti A., Baksi A., Menzel M., Horak P., Paramasivam N., Fazio G., Autry R.J., Fröhling S., Suttorp M., Gertzen C., Gohlke H., Bhatia S., Wadt K., Schmiegelow K., Dugas M., Richter D., Glimm H., Heinäniemi M., Jessberger R., Cazzaniga G., Borkhardt A., Hauer J, & Auer F. (2022). Recurrent Germline Variant in RAD21 Predisposes Children to Lymphoblastic Leukemia or Lymphoma. International Journal of Molecular Sciences, 23(9), 5174.

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Cell lines expressing μ and δ ORs

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μ^βgalOR and μ^βgalOR-δOR expressing U2OS cells were a kind gift from DiscoveRx (Fremont, CA, USA). μ^βgalOR cells expressing μOR tagged with a ProLink/β-galactosidase (βgal) donor (PK) fragment at the C-terminal region and β-arrestin tagged with a complementary βgal activator (EA) fragment were grown in MEM alpha (Life Technologies, Grand Island, NY, USA) containing 10% FBS (Biowest SAS, Nuaille, France), streptomycin-penicillin (Life Technologies), 500 μg/ml geneticin (Life Technologies) and 250 μg/ml hygromycin (Life Technologies). μ^βgalOR-δOR cells expressing wild-type δOR, μOR tagged with the PK fragment at the C-terminal region and β-arrestin tagged with the EA fragment were grown in MEM alpha containing 10% FBS, streptomycin-penicillin, 500 μg/ml geneticin, 250 μg/ml hygromycin and 0.25 μg/ml puromycin (Life Technologies).

Fujita W., Gomes I., Dove L.S., Prohaska D., McIntyre G, & Devi L.A. (2014). Molecular characterization of eluxadoline as a potential ligand targeting mu-delta opioid receptor heteromers. Biochemical pharmacology, 92(3), 448-456.

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Cell lines expressing μ and δ ORs

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Quantifying Lentiviral Barcode Shuffling

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To quantify the extent of barcode shuffling, lentivirus particles of the Neural TF library were produced in an arrayed or pooled manner in 6-well plates as described previously. Multiple wells were transduced at varying titers, the highest transduction titer for which no toxicity or differentiation was seen in the control wells was used for downstream processing. Hygromycin (Thermo Fisher Scientific) selection was started from day 2 onward at a selection dose of 50 μg/ml, medium containing Hygromycin was replaced daily. Cells were harvested 5 days after transduction, spun at 300 rcf for 5 min to obtain cell pellets and genomic DNA extracted.

Parekh U., Wu Y., Zhao D., Worlikar A., Shah N., Zhang K, & Mali P. (2018). Mapping Cellular Reprogramming via Pooled Overexpression Screens with Paired Fitness and Single Cell RNA-Sequencing Readout. Cell systems, 7(5), 548-555.e8.

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Lentiviral Library Transduction Protocol

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Lentiviral libraries 1 + 2 and 3 were used to transduce clones with the 5’ TAG and GAC MS2-adRNA and libraries 4 and 5 + 6 were used to transduce clones with the 3’ TAG and GAC MS2-adRNA stably integrated. Transductions were carried out in duplicates. The lentiviral libraries were mixed with DMEM supplemented with 10% FBS (Thermo Fisher), Hygromycin (Thermo Fisher) at 100 µg/ml, Polybrene Transfection reagent (Millipore) at a concentration of 5 µg/ml and added to the stable clones harboring the MS2-adRNA in a 15 cm dish at 40–50% confluency. To ensure most cells received 0 or 1 ADAR2 variant, cells were transduced at a low MOI of 0.2–0.4. 24 hr post transfections, cells were passaged 1:4 into a new 15 cm dish and grown in DMEM supplemented with 10% FBS (Thermo Fisher) and Hygromycin (Thermo Fisher) at 100 µg/ml. Forty-eight hours post transductions, the growth medium was changed to DMEM supplemented with 10% FBS (Thermo Fisher) and Puromycin (Thermo Fisher) at 3 µg/ml. Seventy-two hours post transduction, fresh growth medium with Puromycin was added to the cells. Ninety-six hours post transductions, the growth media was taken off and cells were washed with PBS and then harvested. Cell pellets were stored at –80°C until RNA extraction. At least 1000× coverage was maintained at all steps of the screen.

Katrekar D., Xiang Y., Palmer N., Saha A., Meluzzi D, & Mali P. (2022). Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity. eLife, 11, e75555.

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Cell Culture and Transfection Protocols

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HEK293T and liver cell lines including Sk-hep1, HepG2, Hep3B, PLC/PRF/5, Huh7, and Mahlavu were cultured as described previously [21 (link)]. HaCaT cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, Taipei, Taiwan) with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific), penicillin (100 U/mL), and streptomycin (100 μg/mL). Lentivirus-infected cells were grown in complete DMEM supplemented with either hygromycin along (100 μg/mL, Thermo Fisher Scientific) or puromycin (1 μg/mL) and hygromycin (100 μg/mL) together. TurboFect Reagent (Fermentas, Hanover, MD, USA) was used for plasmid DNA transfection according to the manufacturers’ instructions.

Chen Y.S., Chang H.S., Hsiao H.H., Chen Y.F., Kuo Y.P., Yen F.L, & Yen C.H. (2021). Identification of Beilschmiedia tsangii Root Extract as a Liver Cancer Cell–Normal Keratinocyte Dual-Selective NRF2 Regulator. Antioxidants, 10(4), 544.

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Generation and Culture of CD79 HEK293T and Sp2/0 Cells

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Ramos, Raji, Daudi, Sp2/0, P3X63, Y3-Ag 1.2.3, HEK293T, and HEK293T-related cells were cultured in IMDM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Cytiva, Marlborough, MA, USA) and 1× pen/strep. CD79 HEK293T and CD79 Sp2/0 cells were generated by cotransduction of HEK293T cells with lentiviral vectors expressing human CD79A with a hygromycin-resistant gene and human CD79B with a puromycin-resistant gene, followed by culture in the presence of hygromycin (800 μg/mL, Thermo Fisher Scientific) and puromycin (2 μg/mL, Thermo Fisher Scientific). Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy normal donors and purchased from the UCLA CFAR Virology Core. PBMCs were cultured in B cell stimulation medium (IMDM supplemented with 10% FBS, 1× pen/strep, 500 IU/mL human interleukin-2 (IL-2; Shenandoah Biotech, Warminster, PA, USA), 50 ng/mL human IL-10 (Shenandoah Biotech), 10 ng/mL human IL-15 (Shenandoah Biotech), 50 nM 2-mercaptoethanol, 1 μg/mL ODN2006 (InvivoGen, San Diego, CA, USA), and 100 ng/mL human soluble CD40 ligand (Shenandoah Biotech

Takano K.A., Wong A.A.L., Brown R., Situ K., Chua B.A., Abu A.E., Pham T.T., Reyes G.C., Ramachandran S., Kamata M., Li M.M.H., Wu T.T., Rao D.S., Arumugaswami V., Dorshkind K., Cole S, & Morizono K. (2024). Envelope protein-specific B cell receptors direct lentiviral vector tropism in vivo. Molecular therapy : the journal of the American Society of Gene Therapy, 32(5).

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Establishing Stable Flp-In™-293 NF-κB Cell Lines

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5 × 10⁶ Flp-In™-293 NF-κB cells were seeded into T75 cell culture flask in 10 ml containing 10% (v/v) FCS and incubated in a humidified incubator at 37 °C under 5% (v/v) CO₂ until cell reached a confluency of about 90%. 2 µg of pcDNA/FRT containing cloned TLR combinations and 18 µg of pOG44 were diluted in 600 µl DMEM. Another 600 µl DMEM with the addition of 40 µl Roti ® -Fect (Carl Roth, Karlsruhe, Germany) was added and the mixture was incubated at room temperature for 20 min and added dropwise to the cells. After 24 h of incubation cells were washed with 10 ml DPBS (Thermo Fisher, A1285601) and 10 ml DMEM containing 10% (v/v) FCS, 1% (v/v) Pen/Strep (10,000 U/ml) was added. 48 h after transfection cells were splitted 1:4 and 300 µg/ml Hygromycin (Thermo Fisher, 10687010) was added to the cell culture medium for antibiotic selection of stably transfected cells. After 24 h, the medium was changed to DMEM (Thermo Fisher, 12491015) with 100 µg/ml Hygromycin and incubation was continued until cell foci appeared. Cell lines were referred to as Flp-In™-293 NF-κB _TLR cells.

Hassan M.I., Keller M., Hillger M., Binder U., Reuter S., Herold K., Telagathoti A., Dahse H.M., Wicht S., Trinks N., Nietzsche S., Deckert-Gaudig T., Deckert V., Mrowka R., Terpitz U., Peter Saluz H, & Voigt K. (2021). The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes. Computational and Structural Biotechnology Journal, 19, 880-896.

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Lentiviral Transduction of H1 Cells

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For viral transduction, on day −1, H1 cells were dissociated to a single cell suspension using Accutase and seeded into Matrigel-coated 6-well plates at a density of 3x10⁵ cells per well in mTeSR containing ROCK inhibitor, Y27632 (10 μM, Tocris). The next day, day 0, cells were approximately 20% confluent. Medium containing Y27632 was replaced with mTeSR1 within 16 hours after plating and cells were allowed to recover for at least 8 hours prior to addition of virus.
Recovered cells were then transduced with lentivirus added to fresh mTeSR containing polybrene (5 μg/ml, Millipore). On day 1, medium was replaced with fresh mTeSR1. Hygromycin (Thermo Fisher Scientific) selection was started from day 2 onward at a selection dose of 50 μg/ml, medium containing Hygromycin was replaced daily.

Parekh U., McDonald D., Dailamy A., Wu Y., Cordes T., Zhang K., Tipps A., Metallo C, & Mali P. (2021). Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas. iScience, 24(10), 103149.

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Lentiviral Transduction of H1 Cells

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For viral transduction, on day -1, H1 cells were dissociated to a single cell suspension using Accutase and seeded into Matrigel-coated 6-well plates at a density of 3x10 5 cells per well in mTeSR containing ROCK inhibitor, Y27632 (10 μM, Tocris). The next day, day 0, cells were approximately 20% confluent. Medium containing Y27632 was replaced with mTeSR1 within 16 hours after plating and cells were allowed to recover for at least 8 hours prior to addition of virus.
Recovered cells were then transduced with lentivirus added to fresh mTeSR containing polybrene (5 μg/ml, Millipore). On day 1, medium was replaced with fresh mTeSR1. Hygromycin (Thermo Fisher Scientific) selection was started from day 2 onward at a selection dose of 50 μg/ml, medium containing Hygromycin was replaced daily.

Parekh U., McDonald D., Dailamy A., Wu Y., Cordes T., Zhang K., Tipps A., Metallo C.M., & Mali P. (2021). Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas.

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