Hemin

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Belgium

Hemin is a lab equipment product produced by Thermo Fisher Scientific. It is a source of heme, an essential cofactor required for various enzymatic reactions and cellular processes. Hemin provides a readily available form of heme for use in biochemical and cell culture applications.

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Close spelling variants of this product

Porcine hemin (2 citations) Remel vitamin K/Hemin solution (1 citations)

35 protocols using hemin

Rat Dermal Fibroblasts: Glucose and AGEs Effects

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Rat dermal fibroblasts (cat. no. CRL-1213; American Type Culture Collection) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) without any supplementary antibiotics in an incubator (37˚C; 5% CO₂). Fibroblasts were cultured in DMEM containing 10% FBS under twelve different conditions (according to the groupings) for 72 h after 24 h of serum deprivation (0.5% FBS). All manipulations were performed in dim lighting, and the plates were wrapped in aluminum foil to protect them from the light. Cells were sub-cultured after they had grown to confluence. All experiments were performed in triplicate.
The cells were divided into the following groups: i) normal glucose (NG) (NG 1.0 g/l; Gibco; Thermo Fisher Scientific, Inc.); ii) NG + Hemin (Hemin 5 µM; MilliporeSigma); iii) NG + chromium mesoporphyrin (CrMP) (CrMP 20 µM; MilliporeSigma); iv) NG + Hemin + CrMP; v) AGEs + NG (AGEs 100 µg/ml; Gibco; Thermo Fisher Scientific, Inc.); vi) AGEs + NG + Hemin; vii) AGEs + NG + CrMP; viii) AGEs + NG + Hemin + CrMP; ix) AGEs + HG (HG 4.5 g/l); x) AGEs + HG + Hemin; xi) AGEs + HG + CrMP; and xii) AGEs + HG + Hemin + CrMP group (9-11 (link)).

Li Q., Liang S., Lai Q., Shen L., Zhang Y, & Guo R. (2021). Heme oxygenase-1 alleviates advanced glycation end product-induced oxidative stress, inflammatory response and biological behavioral disorders in rat dermal fibroblasts. Experimental and Therapeutic Medicine, 22(5), 1212.

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Isolation and Identification of Slow-Growing Bacterial Variants

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Respiratory tract specimens [sputum or oropharyngeal (OP) swabs (Figure S2)] were stored at 4°C after collection and shipped overnight on ice to the CF Foundation-funded Therapeutics Development Network Center for CF Microbiology (TDN-CCFM) at SCH. All respiratory specimens were cultured at the TDN-CCFM’s central laboratory within 48 hr after collection as described previously^{14 (link)}. Culture results from the central study laboratory were not provided to treating physicians. SCVs were defined using a specialized method previously described^{14 (link)}, but with the additional analysis of growth on a laboratory medium (i.e. Mueller-Hinton agar) on which SCVs grow very poorly. SCV auxotrophic testing was performed on Luria-Bertani agar plates with disks impregnated with thymidine (5 μg), hemin (Oxoid), menadione (1.5 μg), or tween 80 (10% solution). Auxotrophy for CO₂ was assessed after growth of SCVs on blood agar plates and Luria-Bertani agar in air compared to 5% CO₂ following 24 hr incubation at 35°C.

Wolter D.J., Onchiri F.M., Emerson J., Precit M.R., Lee M., McNamara S., Nay L., Blackledge M., Uluer A., Orenstein D.M., Mann M., Hoover W., Gibson R.L., Burns J.L, & Hoffman L.R. (2019). Prevalence and clinical associations of Staphylococcus aureus small-colony variant respiratory infection among children with cystic fibrosis: The scvsa multicenter observational study. The Lancet. Respiratory medicine, 7(12), 1027-1038.

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Cultivation of Tannerella forsythia

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Tannerella forsythia ATCC 43037 (American Type Culture Collection, USA) and defined T9SS mutants (see below) were grown in 37 g L⁻¹ of Brain-Heart-Infusion (BHI) liquid media (Oxoid, UK), containing 5 g L⁻¹ yeast extract (Oxoid), 0.5 g L⁻¹ L-cysteine (Sigma, Austria), 2.5 μg mL⁻¹ hemin (Sigma), 2.0 μg mL⁻¹ menadione (Sigma), 10 μg mL⁻¹N-acetylmuramic acid (Carbosynth, UK) and 5% (v/v) horse serum (Life Technologies, Austria), under anaerobic conditions at 37°C for 4-7 days. For cultivation of T. forsythia wild-type and mutants on BHI agar plates (0.8% w/v), the amounts of L-cysteine, hemin, and N-acetylmuramic acid were doubled and plates were incubated under anaerobic conditions in an anaerobic jar (AnaeroJar; Oxoid) at 37°C. Media were supplemented with gentamycin and erythromycin at a concentration of 200 μg mL⁻¹ and 5 μg mL⁻¹, respectively, when appropriate.
Escherichia coli strains were grown under standard conditions in Luria-Bertani (LB) medium supplemented with 100 μg mL⁻¹ ampicillin, when appropriate. P. gingivalis W83 is used as a reference strain for comparison with predicted components of the T9SS in T. forsythia ATCC 43037.

Tomek M.B., Neumann L., Nimeth I., Koerdt A., Andesner P., Messner P., Mach L., Potempa J.S, & Schäffer C. (2014). The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O-glycosylation. Molecular oral microbiology, 29(6), 307-320.

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Preparation of Basal Media for Anaerobic Cultivation

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The basal media was made by mixing yeast extract (Oxoid, Hampshire, UK), Peptone water (BDH, Poole, UK), KH₂PO₄ (BDH), MgSO₄ × 7H₂O NaCl, K₂HPO₄ (BDH), (Fisher Scientific, Loughborough, UK), CaCl₂ × 6H₂O (0.01 g/L), NaHCO₃ (Fisher Scientific), tween-80, hemin (0.05 g/L), vitamin K (10 µL/L), L-cysteine HCl (0.5 g/L), bile salts (0.5 g/L) (Oxoid, Basingstoke, UK) and resazurin solution 0.025 g/100 mL (4 mL/L, pH 7). The obtained solution was heated, cooled, poured into Duran bottles, autoclaved and stored until use.

Obayiuwana O.A., Behrends V., Calle-Patino Y., Barone M., Turroni S., Brigidi P., Costabile A, & Corona G. (2023). Cooking, Digestion, and In Vitro Colonic Fermentation of Nigerian Wholegrains Affect Phenolic Acid Metabolism and Gut Microbiota Composition. International Journal of Molecular Sciences, 24(18), 14111.

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Fungal-Bacterial Biofilm Phenotypic Evaluation

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For phenotypic evaluation, C. albicans SC5314, high biofilm formers (HBF) and low biofilm formers (LBF) [49 (link),50 (link)], and E. faecalis ER5/1, were standardized to 1 × 10⁶ cells/mL for C. albicans and 1 × 10⁷ cells/mL for E. faecalis in Todd-Hewitt broth (THB; Merck UK) supplemented with 10 mM menadione and 10 mg/mL hemin (Thermo Fisher). These were subsequently mixed 1:1 v/v with Roswell Park Memorial Institute (RPMI-1640 [Sigma–Aldrich, Dorset, UK]) (THB:RPMI), a media which has been shown to support the co-culture of C. albicans and bacterial species [51 (link)]. Mono-cultures of C. albicans and co-cultures of C. albicans with E. faecalis were created in a 24 well microtiter plates (Corning Incorporated, Corning, NY, USA) for 24 h in 5% CO₂ at 37 °C. After incubation, biofilms were washed with PBS and stained with 5 μM calcofluor white (Invitrogen, Paisley, UK), a stain which specifically stains the chitin and beta-glucans of fungal cell wall [52 (link)]. Biofilms were incubated in the dark for 20 min and excess stain was washed with sterile water. 2% paraformaldehyde was used for 1 h to fix the stained biofilms which were then imaged using EVOS FL Cell Imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Biofilm biomass of the same biofilms was also quantified using crystal violet stain as previously described [53 ].

Alshanta O.A., Albashaireh K., McKloud E., Delaney C., Kean R., McLean W, & Ramage G. (2022). Candida albicans and Enterococcus faecalis biofilm frenemies: When the relationship sours. Biofilm, 4, 100072.

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Antimicrobial Assay of Proteins Against P. gingivalis

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Porphyromonas gingivalis ATCC 33277 (ATCC, Manassas, VA, USA) were grown anaerobically for at least 24 h (80% N₂, 10% CO₂, 10% H₂) at 37 °C in Todd-Hewitt broth (THB) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.001% hemin (Thermo Fisher Scientific, Waltham, MA, USA) and 0.0001% vitamin K (Thermo Fisher Scientific, Waltham, MA, USA). 20 µM of each of the purified proteins (SCPPPQ1 and ODAM) were exposed in a test tube to 800 µl of a suspension of bacteria at a OD₆₆₀ of 1 (CFU = 10⁹ cells/ml^{13 (link)}) for 2 h at 37 °C. This concentration of P. gingivalis was previously used to experimentally induce periodontitis in mice^{38 (link)}. The protein concentration of 20 μM (160 μg/ml) was selected based on our own prior work showing resistance of SCPPPQ1 to degradation by P. gingivalis^{13 (link)}. Also, it represented a concentration within the range used in other studies looking at the effects of antimicrobial effects of peptides^{17 (link),18 (link),34 (link),42 (link),43 (link)}. The mixture was then sampled for fluorescence microscopy, SEM and TEM characterization. As a negative control, buffer without the proteins was incubated with P. gingivalis under the same conditions.

Fouillen A., Mary C., Ponce K.J., Moffatt P, & Nanci A. (2021). A proline rich protein from the gingival seal around teeth exhibits antimicrobial properties against Porphyromonas gingivalis. Scientific Reports, 11, 2353.

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Cell Culture and Differentiation Protocols

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K562 and TF-1 human erythroleukemia and Jurkat human T-cell leukemia cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). They were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS; Mediatech, Herndon, VA), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM l-glutamine, and 4.5 g/l glucose. Jurkat and TF-1 culture media contained 1 mM sodium pyruvate, and TF-1 cells were cultured in the presence of 2 ng/ml granulocyte-macrophage colony-stimulating factor. SH-SY5Y human neuroblastoma cells were also purchased from ATCC. They were cultured in 1:1 mixture of MEM with nonessential amino acids and Ham's F12 medium containing 10% FBS. They were maintained at 37°C in a humidified 95% air, 5% carbon dioxide incubator. For K562 erythroid differentiation, exponentially growing cells were exposed to hemin (Fluka-Sigma-Aldrich, St. Louis, MO). hemin and sodium arsenite (Thermo Fisher Scientific, Waltham, MA) were dissolved in 100 μM NaOH and distilled water, respectively. t-BHQ (Sigma-Aldrich, St. Louis, MO) was dissolved in dimethyl sulfoxide for 1 M solution and further diluted to 10 mM with distilled water before addition to the culture media. Sulforaphane (Sigma-Aldrich) was also dissolved in dimethyl sulfoxide for 100 mM solution.

Miyazawa M, & Tsuji Y. (2014). Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene. Molecular Biology of the Cell, 25(13), 2116-2127.

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Purification of Mfa1 and FimA Fimbriae from P. gingivalis Mutants

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Mfa1 and FimA fimbriae were purified from P. gingivalis mutant JI-1 (fimA deleted) [36 (link),37 (link)] and SMF-1 (mfa1 deleted) [38 (link),39 (link)] derived from ATCC 33277. JI-1 and SMF-1 cells were cultured under anaerobic conditions at 37 °C on Brucella HK agar medium (KYOKUTO, Tokyo, Japan) prepared by mixing 5% [volume/volume (v/v)] laked rabbit blood, 2.5 μg/mL hemin (Sigma-Aldrich, St. Louis, MO, USA), 5 μg/mL menadione (Sigma-Aldrich), and distilled water. Liquid medium was prepared by mixing trypticase soy broth (Thermo Fisher Scientific), 0.25% yeast extract (Thermo Fisher Scientific), and distilled water, followed by sterilization and the addition of 2.5 μg/mL hemin and 5 μg/mL menadione.

Suzuki Y., Kikuchi T., Goto H., Takayanagi Y., Kawamura S., Sawada N., Naiki Y., Kondo H., Hayashi J.I., Hasegawa Y, & Mitani A. (2022). Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells. International Journal of Molecular Sciences, 23(23), 15293.

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Enriched Todd Hewitt Broth for Dual Culture

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Todd Hewitt broth (THB, Sigma-Aldrich) was prepared and supplemented with 10 µM menadione and 4 mg/mL hemin (ThermoFisher) and mixed 1:1 with Roswell Park Memorial Institute media (RPMI). Referred to as sTHB from herein. A similar media has been described elsewhere and has been shown to enable the growth of both fungi and bacteria (Montelongo-Jauregui et al., 2016 (link)).

Short B., Delaney C., McKloud E., Brown J.L., Kean R., Litherland G.J., Williams C., Martin S.L., MacKay W.G, & Ramage G. (2021). Investigating the Transcriptome of Candida albicans in a Dual-Species Staphylococcus aureus Biofilm Model. Frontiers in Cellular and Infection Microbiology, 11, 791523.

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Anaerobic Cultivation of Bacterial Strains

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Bacterial strains (Additional file 1: Table S1) were from either the American Type Culture Collection (ATCC) or from the Collection du Laboratoire de Recherche de Technologie Laitière, Institut National de la Recherche Agronomique, Rennes, France. Colonies were grown on Brucella agar with 5% sheep’s blood, hemin, and vitamin K (Thermo Fisher Scientific, Remel Products, Lenexa, KS, USA) at 30 °C for 5-7 days under anaerobic conditions in a sealed anaerobic box with oxygen-absorbing, carbon dioxide-generating AnaeroPack-Anaero® sachets (Mitsubishi Gas Chemical Co., Inc. [MGC], Tokyo, Japan). Liquid cultures inoculated from single colonies were grown anaerobically at 30 °C to mid-log phase (OD₆₀₀ = 0.4-0.7) in yeast extract sodium lactate (YEL) media, containing (per Liter), 10 g pancreatic digest of casein, 10 g yeast extract, 10 g sodium lactate, 2.5 g KH₂PO₄, 5 mg MnSO₄ [20 (link)].

Cheng L., Marinelli L.J., Grosset N., Fitz-Gibbon S.T., Bowman C.A., Dang B.Q., Russell D.A., Jacobs-Sera D., Shi B., Pellegrini M., Miller J.F., Gautier M., Hatfull G.F, & Modlin R.L. (2018). Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages. BMC Microbiology, 18, 19.

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