Hygromycin b

Manufactured by Formedium

Hygromycin B is a laboratory reagent used as a selection marker in cell culture and genetic engineering applications. It is an antibiotic that inhibits protein synthesis in eukaryotic cells, enabling the identification and selection of cells that have been successfully transformed with a gene of interest.

Automatically generated - may contain errors

Lab products found in correlation

12 protocols using hygromycin b

Fluorescent Mycobacterial Strains for Research

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

WT M. marinum M strains (ATCC #BAA-535) expressing tdTomato, tdKatushka, or EBFP2 under the msp12 promoter (Takaki et al., 2013 (link)) were grown under hygromycin B (Formedium) selection in 7H9 Middlebrook medium (Difco) supplemented with oleic acid, albumin, dextrose, and Tween-80 (Sigma) (Takaki et al., 2013 (link)).
M. tuberculosis ΔleuD ΔpanCD double auxotroph expressing mCherry or GFP under control of the msp12 promoter were grown under hygromycin B (Formedium) and kanamycin selection in Middlebrook 7H9 broth (Difco) supplemented with oleic acid, albumin, dextrose, catalase, Tween-80 (Sigma), 0.05 mg/ml L-leucine and 0.024 mg/ml calcium pantothenate (Sigma) (Sampson et al., 2011 (link)).

Roca F.J., Whitworth L.J., Redmond S., Jones A.A, & Ramakrishnan L. (2019). TNF Induces Pathogenic Programmed Macrophage Necrosis in Tuberculosis through a Mitochondrial-Lysosomal-Endoplasmic Reticulum Circuit. Cell, 178(6), 1344-1361.e11.

+ Open protocol

+ Expand

Generating Stable Cell Lines with CD8 Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols

To generate stable cell lines, HeLa TRex Flp-In host cells (provided by Stephen Taylor, University of Manchester, Manchester, UK) were transfected with CD8 variants. Stably transfected cells were selected using hygromycin B (ForMedium) and blasticidin (InvivoGen). Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% non-essential amino acids at 37°C and under 8% CO₂. Experiments were performed after inducing expression with 1 µg/ml tetracycline for 16–20 h unless otherwise stated. For transient transfections, HeLa cells were transfected using Lipofectamine LTX (Invitrogen) and analysed after 16–20 h.

Briant K., Koay Y.H., Otsuka Y, & Swanton E. (2015). ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues. Journal of Cell Science, 128(22), 4112-4125.

+ Open protocol

+ Expand

Yeast Strain Generation and Genetic Manipulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Yeast strains were created either by random spore digestion or by tetrad dissection, as described previously (Forsburg and Rhind, 2006 (link)). Gene deletion and tagging were performed by the PCR-based method using the pFA6a series of plasmids (Hentges et al., 2005 (link)), and yeast transformation was carried out by the lithium acetate method. Culture media were purchased from Formedium (www.formedium.com), and the drugs G418, nourseothricin, and hygromycin B were purchased from Formedium, Werner Bioagents (www.webioage.de), and Sigma Aldrich (www.sigmaaldrich.com), respectively. For live-cell imaging, cells were cultured in Edinburgh minimal medium supplied with adenine, leucine, uracil, histidine, and lysine (0.225 g/l each), and for biochemistry, cells were cultured in yeast extract (YE) medium containing the five supplements. The yeast strains used in this study are listed in Supplemental Table S1.

Shen J., Li T., Niu X., Liu W., Zheng S., Wang J., Wang F., Cao X., Yao X., Zheng F, & Fu C. (2019). The J-domain cochaperone Rsp1 interacts with Mto1 to organize noncentrosomal microtubule assembly. Molecular Biology of the Cell, 30(2), 256-267.

+ Open protocol

+ Expand

MDM2 Protein Expression in Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

Unless otherwise stated, cell culture reagents were purchased from Gibco (ThermoFisher Scientific). A549 cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM:F12) with HEPES, supplemented with 10% foetal bovine serum (JRH Biosciences #12003C or Sigma-Aldrich #F9423) and 2 mM GlutaMAX™ or 2 mM L-Glutamine. HEK-293 cells were cultured in DMEM supplemented with 10% FBS (Sigma-Aldrich #F9423) and 2mM L-glutamine. MCF10A cells were cultured in DMEM:F12 containing 5% horse serum (#16050-122), 20 ng/mL epidermal growth factor (EGF, Peprotech), 0.5 mg/mL hydrocortisone (Sigma-Aldrich #H-0888), 100 ng/mL cholera toxin (Sigma-Aldrich #C-8052) and 10 μg/mL insulin (available from pharmacy). The Flp-In™ T-Rex™ U2OS cells were cultured in DMEM supplemented with 10% FBS and 1 μg/mL Blasticidin (Melford). For expression of MDM2 protein, the cDNA for the human full-length MDM2 sequence was cloned into the pcDNA5 vector (ThermoFisher Scientific) to enable expression of the proteins with an N-terminal 2xFLAG-PreScission protease site-His6 (FLAG) tag. The plasmid was then transfected into the cells for stable integration into the host genome and selected using Hygromycin B (Formedium) according to the manufacturers’ instructions.

Hannan K.M., Soo P., Wong M.S., Lee J.K., Hein N., Poh P., Wysoke K.D., Williams T.D., Montellese C., Smith L.K., Al-Obaidi S.J., Núñez-Villacís L., Pavy M., He J.S., Parsons K.M., Loring K.E., Morrison T., Diesch J., Burgio G., Ferreira R., Feng Z.P., Gould C.M., Madhamshettiwar P.B., Flygare J., Gonda T.J., Simpson K.J., Kutay U., Pearson R.B., Engel C., Watkins N.J., Hannan R.D, & George A.J. (2022). Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway. Cell Reports, 41(5), 111571.

+ Open protocol

+ Expand

Generating Stable HeLa TRex Flp-In Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

Stable HeLa TRex Flp-In cells expressing CD8 chimeras were generated by transfecting parental TRex HeLa cells (provided by Steven Taylor, University of Manchester) using Lipofectamine LTX (Invitrogen) following manufacturer’s instructions. Transfected cells were selected using 100 μg/ml hygromycin B (ForMedium) and 4 μg/ml blasticidin (InvivoGen). Once established, cell lines were maintained in complete DMEM (DMEM (Sigma) supplemented with 10% FBS, 2 mM L-glutamine and 1% non-essential amino acids) at 37°C, 8% CO₂. All experiments were performed after inducing expression with 1 μg/ml tetracycline for 16–20 hours unless otherwise stated. For siRNA transfections, cells were seeded at 25,000 cells / well in a 12 well dish. The following day, cells were transfected with INTERFERin (Polyplus Transfection), using a final concentration of 20 nM siRNA duplex. Experiments were performed 48–72 hours post-transfection. Rer1 targeting siRNA oligo 1 sequence: 5’-UAUCAGUCCUGGCUAGAC-3’; Rer1 siRNA oligo 2 sequence: 5’-UGCGAGUUACAGAAUGUCUGA-3’. Calnexin siRNA sequence: 5’-UCAUCAUCGGUAUCGUCUU-3’

Briant K., Johnson N, & Swanton E. (2017). Transmembrane domain quality control systems operate at the endoplasmic reticulum and Golgi apparatus. PLoS ONE, 12(4), e0173924.

+ Open protocol

+ Expand

Yeast Experimental Conditions Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

All general growth conditions and yeast manipulations were performed as previously described (Moreno et al., 1991 (link); Forsburg and Rhind, 2006 (link)). The relevant genotypes and the source of the utilized strains are listed in Supplementary Table 1. All experiments were performed with cells from cultures growing exponentially. Normally, cells were cultured in YES (Yeast Extract with Supplements; 0.5% yeast extract, 3% glucose, 225 mg/l adenine sulfate, histidine, leucine, uracil, and lysine). When required, cells were grown in EMM2 (Edinburgh Minimal Medium) (Moreno et al., 1991 (link)) with supplements. For drop-test analyses to assess sensitivity to ionic stress, 3 × 10⁴ cells and serial 1:4 dilutions were inoculated on YES plates supplemented or not with different salts and incubated at 32°C for 3 days. When the assays were performed on minimal EMM2 plates, KNO₃ was preferred to KCl for technical reasons, and the plates were incubated for 5 days. Geneticin (G418, Formedium), hygromycin B (Formedium), and nourseothricin (Werner BioAgents) were used at 120, 400, and 50 μg/ml, respectively.

Moro S., Moscoso-Romero E., Poddar A., Mulet J.M., Perez P., Chen Q, & Valdivieso M.H. (2021). Exomer Is Part of a Hub Where Polarized Secretion and Ionic Stress Connect. Frontiers in Microbiology, 12, 708354.

+ Open protocol

+ Expand

Synthetic Media for Yeast Cultivation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Synthetic media used in this study contains 6.7 g/l yeast nitrogen base with ammonium sulfate (Conda Pronadisa #1545) and either 2% glucose, 2% ethanol, 3% glycerol, or 0.2% oleic acid (Sigma) +0.1% Tween 80, with complete amino acid mix (oMM composition, Hanscho et al, 2012 (link)), unless written otherwise; when Hygromycin or Geneticin antibiotics were used, media contains 0.17 g/l yeast nitrogen base without Ammonium Sulfate (Conda Pronadisa #1553) and 1 g/l of monosodium glutamic acid (Sigma‐Aldrich #G1626) instead of yeast nitrogen base with ammonium sulfate. When mentioned, 500 mg/l Hygromycin B (Formedium), 500 mg/l Geneticin (G418; Formedium), and 200 mg/l Nourseothricin (WERNER BioAgents “ClonNat”) were used.

Yifrach E., Holbrook‐Smith D., Bürgi J., Othman A., Eisenstein M., van Roermund C.W., Visser W., Tirosh A., Rudowitz M., Bibi C., Galor S., Weill U., Fadel A., Peleg Y., Erdmann R., Waterham H.R., Wanders R.J., Wilmanns M., Zamboni N., Schuldiner M, & Zalckvar E. (2022). Systematic multi‐level analysis of an organelle proteome reveals new peroxisomal functions. Molecular Systems Biology, 18(9), e11186.

+ Open protocol

+ Expand

Yeast Strains and Growth Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols

S. cerevisiae strains used in this study are listed in Table S4. Most strains are based on BY4741 and BY4742 (Brachmann et al., 1998 (link)), W303-1A (Thomas and Rothstein, 1989 (link)) and the yeast deletion collection (Giaever et al., 2002 (link)). Double mutants were generated by crossing haploid single mutants using standard procedures and all double mutants were confirmed by PCR. Yeast cells were grown in minimal synthetic complete (SC) medium (0.67% yeast nitrogen base) supplemented with auxotrophic requirements and 2% glucose as a carbon source or in rich yeast peptone dextrose (YPD) medium. Growth assays on solid agar were carried out as previously described (Wysocki et al., 2004 (link)). Sodium arsenite (NaAsO₂), 1,10-phenantroline (both from Sigma-Aldrich) and hygromycin B (Formedium) were added to the cultures at the indicated concentrations.

Andersson S., Romero A., Rodrigues J.I., Hua S., Hao X., Jacobson T., Karl V., Becker N., Ashouri A., Rauch S., Nyström T., Liu B, & Tamás M.J. (2021). Genome-wide imaging screen uncovers molecular determinants of arsenite-induced protein aggregation and toxicity. Journal of Cell Science, 134(11), jcs258338.

+ Open protocol

+ Expand

Generating Transgenic C. elegans Lines

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Strains were maintained under standard conditions unless otherwise indicated (Brenner, 1974 (link); Stiernagle, 2006 (link)).
Transgenic worms were generated by biolistic bombardment using hygromycin B as a selection marker (Greiss and Chin, 2011 (link); Radman et al., 2013 (link); Davis and Greiss, 2018 (link)) in either N2 or smg-2(e2008) genetic background. The smg-2(e2008) background lacks a functional nonsense-mediated decay machinery and was used to increase levels of reporter mRNA (Greiss and Chin, 2011 (link)). Gamma-irradiation to generate the integrated line SGR56 from SGR55 was carried out by Michael Fasseas (Invermis/Magnitude Biosciences). After integration, the line was backcrossed into N2 and subsequently maintained on standard NGM without added hygromycinB. SGR56 was backcrossed twice, SGR96 was backcrossed four times. All non-integrated lines were maintained on NGM supplemented with hygromycin B (0.3 mg/ml; Formedium). Strains used in this paper are listed in the key resources table.

Davis L., Radman I., Goutou A., Tynan A., Baxter K., Xi Z., O'Shea J.M., Chin J.W, & Greiss S. (2021). Precise optical control of gene expression in C elegans using improved genetic code expansion and Cre recombinase. eLife, 10, e67075.

+ Open protocol

+ Expand

Synthetic Media Formulations for Research

Check if the same lab product or an alternative is used in the 5 most similar protocols

Synthetic media used in this study contains 6.7 g/L yeast nitrogen base with ammonium sulfate (Conda Pronadisa #1545) and 2% glucose, with complete amino acid mix (oMM composition, Hanscho et al. 2012) , unless written otherwise. When Hygromycin or Geneticin antibiotics were used, media contained 0.17 g/L yeast nitrogen base without ammonium sulfate (Conda Pronadisa #1553) and 1 g/L of monosodium glutamic acid (Sigma-Aldrich #G1626) instead of yeast nitrogen base with ammonium sulfate. When mentioned, 500 mg/L Hygromycin B (Formedium), 500 mg/L Geneticin (G418) (Formedium), and 200 mg/L Nourseothricin (Silcol Scientific Equipment LTD) were used.

Yifrach E., Rudowitz M., Cruz-Zaragoza L.D., Tirosh A., Gazi Z., Peleg Y., Kunze M., Eisenstein M., Schliebs W., Schuldiner M., Erdmann R, & Zalckvar E. (2023). Determining the targeting specificity of the selective peroxisomal targeting factor Pex9. Biological chemistry, 404(2-3).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!