Adult bovine serum

Manufactured by Merck Group

Sourced in United States

Adult bovine serum is a cell culture reagent derived from the blood of adult cattle. It is a complex mixture of proteins, growth factors, and other biomolecules that supports the growth and maintenance of various cell types in in vitro cell culture applications.

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Lab products found in correlation

Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Ascorbic acid, by Fujifilm (1 mentions) Opti mem 1 medium, by Thermo Fisher Scientific (1 mentions) Lipofectamine, by Thermo Fisher Scientific (1 mentions) Plus reagent, by Thermo Fisher Scientific (1 mentions) L cysteine, by Merck Group (1 mentions) Human apo transferrin, by Merck Group (1 mentions) Infinite m200 pro, by Tecan (1 mentions) Infinite m200, by Tecan (1 mentions) Axio vert a1 inverted microscope, by Zeiss (1 mentions) Biocoat matrigel invasion chamber, by Corning (1 mentions) Hrp conjugated anti mouse igm, by Santa Cruz Biotechnology (1 mentions) Pyranose oxidase, by Merck Group (1 mentions) Creatine phosphokinase, by Merck Group (1 mentions) Creatine phosphate, by Merck Group (1 mentions) Catalase, by Merck Group (1 mentions) Methylcellulose, by Merck Group (1 mentions) 50 ml culture flasks, by Greiner (1 mentions) Mem vitamin solution, by Thermo Fisher Scientific (1 mentions)

19 protocols using adult bovine serum

Immunofluorescence Analysis of Small Intestine

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Proximal, mid and distal segments of the SI were fixed with 4% paraformaldehyde (PFA) from 3 h to overnight at 4 °C, embedded in OCT and kept at −80 °C until sectioning. Sections (7 µm thick) were prepared with a cryotome (Cryostat CM 3050S).
Images from Hematoxyline QS (Vector) stained were acquired using NDP Zoomer Digital Pathology (Hamamatsu) and subsequently analysed in NDP.view2 software.
To perform immunofluorescence, sections were blocked and permeabilized in 10% adult bovine serum (Sigma), 5% skim milk (Sigma) and 0.3% Triton X-100 in PBS (blocking buffer) for at least 1 h at 4 °C. Primary antibodies (listed in Supplementary Table 3) were incubated overnight in blocking buffer at 4 °C. Alexa-Fluor-Conjugated secondary antibodies (indicated in Supplementary Table 3) were incubated for 1–2 h at room temperature in 10% adult bovine serum and 0.1% bovine serum albumin (BSA) (Sigma) in PBS. Diamidino-2-phenylindole dihydrochloride (DAPI; 1 µM; Sigma) was used to counterstain nuclei in the indicated experiments. Images were acquired using laser-scanning confocal microscopes (Leica TSC SP8 and Zeiss LSM800). All images were subsequently analysed with Fiji software.

Maimets M., Pedersen M.T., Guiu J., Dreier J., Thodberg M., Antoku Y., Schweiger P.J., Rib L., Bressan R.B., Miao Y., Garcia K.C., Sandelin A., Serup P, & Jensen K.B. (2022). Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling. Nature Communications, 13, 715.

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Purification of Actin, Myosin, and Gelsolin

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Actin and skeletal muscle myosin were purified from rabbit skeletal muscle^{47 (link),48 (link)}. No rabbits were directly involved in the study. Monomeric actin (G-actin) was stored in G-buffer at 4 °C. Heavy meromyosin (HMM) was prepared by dialyzing ground rabbit skeletal muscle against the M-buffer at 4 °C. Biotinylated HMM (bHMM) was prepared by incubating HMM with NHS-biotin in a 1:10 molar ratio in DMSO at room temperature for 10 min (Supplementary Information). Gelsolin was purified from adult bovine serum (Sigma-Aldrich) (Supplementary Information). Streptavidin was purchased from Thermo Fisher Scientific. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich.

Hsu C.P., Sciortino A., de la Trobe Y.A, & Bausch A.R. (2022). Activity-induced polar patterns of filaments gliding on a sphere. Nature Communications, 13, 2579.

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Efficient Transfection and Selection of Entamoeba histolytica Transformants

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Transfection of trophozoites and selection and maintenance of transformants were performed based on the method of Nozaki et al34 (link). Briefly, liposomes containing pEhEx/AS-Myc were made with 5 μg plasmid, Lipofectamine^® and Plus^TM reagent (both Life Technologies). The liposome was mixed into OPTI-MEM I medium (Life Technologies) with 5 mg/ml L-cysteine (Sigma) and 1 mg/ml ascorbic acid (Wako, Osaka, Japan). Approximately 5.0 × 10⁵ trophozoites were incubated with the medium containing the liposome at 37 °C for 5 h. After incubation, trophozoites were transferred into culture test tubes with warmed YIMDHA-S medium supplemented with 15% adult bovine serum (Sigma). Drug selection by G418 was begun from 24 h later and medium was replaced by fresh medium with G418 every 24 h.

Kazama M., Ogiwara S., Makiuchi T., Yoshida K., Nakada-Tsukui K., Nozaki T, & Tachibana H. (2017). Behavior of DNA-lacking mitochondria in Entamoeba histolytica revealed by organelle transplant. Scientific Reports, 7, 44273.

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Bacterial Growth Assay in Complex Media

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A V. campbellii overnight culture was diluted 1:100 into Kim-Epstein (KE) medium (10 mL) (56 (link)) or lysogeny broth containing 3.5% (wt/vol) NaCl (LB35) medium (60 mL) and grown until early exponential growth phase (optical density at 600 nm [OD₆₀₀] 0.5, KE medium) or early stationary phase (OD₆₀₀ 5.0, LB35). The KE medium was modified as follows: The pH was adjusted to 7.6 with the corresponding phosphate buffer, the salt concentration was increased to 2% (wt/vol) NaCl, and FeSO₄ was omitted. Compounds were added from DMSO stocks to 50 μM into a clear flat-bottom 96-well plate. Next, 200 μL bacterial culture previously diluted to OD₆₀₀ 0.01 in KE medium + 20 mM NaHCO₃ or in KE medium + 20 mM NaHCO₃ supplemented with 100 μg/mL human apo-transferrin (Sigma-Aldrich) or to OD₆₀₀ 0.005 in LB35 or in LB35 supplemented with 30% (vol/vol) of adult bovine serum (Sigma-Aldrich) was added. The plates were incubated in an Infinite M200 Pro plate reader (Tecan) at 30 °C with continuous shaking (KE medium) or with 20 s shaking every 5 min (LB35 medium). The optical density at 600 nm was measured every 10 to 30 min. Blank values (only medium) were subtracted from data values, and data were plotted using GraphPad Prism.

Weigert Muñoz A., Hoyer E., Schumacher K., Grognot M., Taute K.M., Hacker S.M., Sieber S.A, & Jung K. (2022). Eukaryotic catecholamine hormones influence the chemotactic control of Vibrio campbellii by binding to the coupling protein CheW. Proceedings of the National Academy of Sciences of the United States of America, 119(10), e2118227119.

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Catecholamine-Mediated Bacterial Growth Assay

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For the bacterial growth assays, V. harveyi was grown overnight in LB₃₅ broth at 28°C. After that, the culture was re-inoculated at a concentration of 10² CFU/ml into fresh LB₃₅ broth containing 30% (v/v) adult bovine serum (Sigma–Aldrich), with and without 50 μM NE or Dopa. Additionally, different concentrations of the catecholamine receptor antagonists were added in conjunction with the catecholamines to determine whether they could neutralize catecholamine-induced growth responses. The cultures were grown in 200 μl volumes in 96-well plates at 28°C for 48 h, and the turbidity at 600 nm was monitored every hour using a Multireader machine (Infinite M200, TECAN, Austria). Growth curves were determined for three independent cultures, and the growth rate of the exponentially growing cultures was calculated. The statistical significance of specific growth rate was determined using an independent samples t-test.

Yang Q., Anh N.D., Bossier P, & Defoirdt T. (2014). Norepinephrine and dopamine increase motility, biofilm formation, and virulence of Vibrio harveyi. Frontiers in Microbiology, 5, 584.

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Invasion Assay of E. histolytica

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This was performed as described previously (Emmanuel et al., 2015 (link)). Briefly, 75,000 E. histolytica trophozoites were pre-incubated for 3 h with 0.5% DMSO, 14 and 25 μM of tipifarnib and after 3 h, cells were re-suspended in serum-free TYI medium and loaded in the upper chamber of a Corning BioCoat Matrigel Invasion Chamber (Corning). The lower chamber contained TYI medium supplemented with 10% adult bovine serum (Sigma-Aldrich). The Matrigel Invasion Chamber was incubated at 37°C for 48 h in a GasPak EZ gas-generating anaerobe pouch system. At the end of incubation, images of trophozoites in the lower chamber were captured using a 10 × objective lens fitted in a Zeiss Axiovert A1 inverted microscope. Trophozoites that had migrated into the lower chamber were also counted using a hemocytometer. The data were obtained from three independent experiments each performed in triplicate and the percentage invasion of trophozoites was calculated and plotted by using GraphPad Prism.

Probst A., Nguyen T.N., El-Sakkary N., Skinner D., Suzuki B.M., Buckner F.S., Gelb M.H., Caffrey C.R, & Debnath A. (2019). Bioactivity of Farnesyltransferase Inhibitors Against Entamoeba histolytica and Schistosoma mansoni. Frontiers in Cellular and Infection Microbiology, 9, 180.

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ELISA for Recombinant Antibody Quantification

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We developed an ELISA system to monitor the amount of recombinant antibodies in various samples. Maxisorp plates were coated with 100 μl of 0.5 μg/ml anti-mouse kappa light chain (Sigma-Aldrich, St. Louis, MO). Plates were blocked at RT for 3 hours with TBS containing 10% adult bovine serum (Sigma), 5% sucrose and 0.05% sodium azide, dried at RT for 2 hours, stored at 4 °C and used with 1 month. Antibody samples were diluted in TBS containing 2% adult bovine serum and 0.01% Tween 20. Diluted samples were incubated with the plates at RT for 1 hour and washed 4 times with TBS containing 0.05% Tween 20. The plates were incubated with 1:4,000 dilution of HRP-conjugated anti-mouse IgM (Santa Cruz Biotechnology, Santa Cruz, CA) or 1: 20,000 anti-mouse IgG (Nacalai Tesque, Kyoto, Japan) at RT for 1 hour. After washing, color was developed with TMB. Reactions were stopped with 0.5 M HCl and the optical density was read with plate reader at 450 nm. Antibody amounts were estimated with the built-in software of the plate reader using appropriate Ig as standard ranging from 3.125 to 200 ng/ml.

Teye K., Hashimoto K., Numata S., Ohta K., Haftek M, & Hashimoto T. (2017). Multimerization is required for antigen binding activity of an engineered IgM/IgG chimeric antibody recognizing a skin-related antigen. Scientific Reports, 7, 8212.

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Kinesin, Microtubule, and Actin Assay

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Biotin–kinesin and MTs stabilized with GMP-CPP (Guanosine-5'-[(α,β)-methyleno]triphosphate) were obtained from Brandeis University’s National Science Foundation Materials Research Science and Engineering Center and stored at –80 °C. Kinesin, once thawed, was kept on ice, while MTs were kept at room temperature. Actin was purified from rabbit skeletal muscle as described previously^{9 (link)}. No rabbits were directly involved in the study. Monomeric actin was stored at 4 °C in the so-called G-Buffer (2 mM Tris, 0.2 mM ATP, 0.2 mM CaCl₂, 0.2 mM DTT and 0.005% NaN₃ at pH 8.0). Gelsolin was purified from adult bovine serum (Sigma-Aldrich). Alexa Fluor 488 phalloidin, streptavidin and Texas Red DHPE dye were purchased from Thermo Fisher. The remaining lipids (l-α-phosphatidylcholine (egg PC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl(polyethylene glycol)-2000]) (DSPE–PEG(2000)–biotin, where 2,000 is the PEG molecular weight)) were purchased from Avanti and stored in chloroform. Methylcellulose, creatine phosphate, creatine phosphokinase, catalase and pyranose oxidase were purchased from Sigma.

Sciortino A., Neumann L.J., Krüger T., Maryshev I., Teshima T.F., Wolfrum B., Frey E, & Bausch A.R. (2022). Polarity and chirality control of an active fluid by passive nematic defects. Nature Materials, 22(2), 260-268.

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Purification of Actin and Skeletal Myosin

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Actin and skeletal muscle myosin were purified from rabbit skeletal muscle. No rabbits were directly involved in the study. Monomeric actin was stored at 4 °C in G-Buffer (2 mM Tris, 0.2 mM ATP, 0.2 mM CaCl2, 0.2 mM DTT, and 0.005% NaN3 at pH 8.0). HMMs were prepared by dialyzing ground rabbit skeletal muscle against the Myosin-buffer (0.6 M KCl, 10 mM KH₂PO₄, and 2 mM DTT) at 4 °C (77 ). Gelsolin was purified from adult bovine serum (Sigma Aldrich) (SI Appendix). Alexa Fluor 488 phalloidin, dark phalloidin, and streptavidin are purchased from Thermo Fisher. Lipids (Egg PC, DSPE-PEG [2000] Biotin, Texas Red DHPE) are purchased from Avanti.

Sciortino A, & Bausch A.R. (2021). Pattern formation and polarity sorting of driven actin filaments on lipid membranes. Proceedings of the National Academy of Sciences of the United States of America, 118(6), e2017047118.

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Culturing E. histolytica Trophozoites

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E. histolytica strain HM-1:IMSS trophozoites were maintained in 50ml culture flasks (Greiner Bio-One) containing TYI-S-33 media, 10% heat-inactivated adult bovine serum (Sigma), 1% MEM Vitamin Solution (Gibco), supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Omega Scientific) [14 (link)].

Sauvey C., Ehrenkaufer G., Shi D., Debnath A, & Abagyan R. (2021). Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds. PLoS Neglected Tropical Diseases, 15(2), e0008425.

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