One million of epimastigote forms of T. cruzi MHOM/BR/00/Y (DTU II) [63 ] were grown in Liver Infusion Tryptose (LIT) medium supplemented with 10% bovine serum albumin (Eurobio, Les Ulis, France) at 26 °C; after 96 h of culture, 2 × 108 epimastigotes were obtained. In the meantime, Green Monkey renal fibroblast-like cells (Vero cells; ATCC CCL-81, Manassas, VA, USA) were grown in Dulbecco’s modified eagle medium (DMEM) (Eurobio, Les Ulis, France) supplemented with 10% bovine serum albumin (Eurobio, Les Ulis, France), 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.01 M hepes (Eurobio, Les Ulis, France) at 37 °C in a humid atmosphere with 5% CO2. After reaching semiconfluence, the Vero cells were incubated for 10 h with 3 × 107T. cruzi trypomastigotes MHOM/BR/00/Y (DTU II) [63 ], and the parasites were recovered at 96 h postinfection. Trypomastigotes with three cyclic passages in Vero cells were used in subcellular location assays. Similarly, 106 promastigotes of Leishmania major MHOM/IL/81/Friedlin strain [64 (link)] were grown in Schneider’s insect medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 20% bovine serum albumin (Eurobio, Les Ulis, France), and 1 μg/mL of 6-biopterin (Sigma-Aldrich, St. Louis, MO, USA) at 26 °C; after 72 h of culture 8 × 106 promastigotes in logarithmic growth phase were obtained.
Bovine serum albumin (bsa)
Manufactured by Eurobio Scientific
Sourced in France
Bovine serum albumin (BSA) is a widely used laboratory reagent derived from the serum of bovine (cattle) origin. It is a protein that serves as a stabilizer, blocking agent, and protein standard in various biological applications.
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Lab products found in correlation
Penicillin, by Eurobio Scientific (2 mentions)
Streptomycin, by Eurobio Scientific (2 mentions)
Chemiluminescence detection system, by GE Healthcare (2 mentions)
Gradient polyacrylamide gel, by Bio-Rad (2 mentions)
Imagescanner 3, by GE Healthcare (2 mentions)
Acrylamide, by Eurobio Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Hepes, by Eurobio Scientific (2 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Ly294002, by Thermo Fisher Scientific (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Ecl system, by Cytiva (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Gelatin solution, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer pack, by Bio-Rad (1 mentions)
Recombinant human il 6, by R&D Systems (1 mentions)
Anti collagen type 1 antibody, by Merck Group (1 mentions)
12 protocols using bovine serum albumin (bsa)
1
Cultivation of T. cruzi and L. major
2
Overcoming PCR Inhibition in Vegetable Matrices
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In other series of experiments to test the inhibition of the PCR assay by the vegetable matrix, we inoculated 50 ml of saline solution at 0.9% containing 100 Giardia cysts in the polypropylene bags containing 35 g of lettuce or cabbage leaves and DNA extraction was performed. In this group of samples, we evaluated the effect of adding three different concentrations of bovine serum albumin (BSA, Eurobio, France) to the PCR mix. After this, the BSA optimal concentration to reduce PCR inhibition was used to evaluate the reproducibility, in three separate experiments, with cabbage and lettuce samples inoculated with 10 Giardia cysts.
3
Cellular Oxidative Stress Assays
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Chemicals were purchased from the following manufacturers: Dulbecco Modified Eagle’s Medium/HAM F12 from Gibco Cell Culture, Thermo Fisher Scientific—Invitrogen, (Carlsbad, CA, USA); penicillin, streptomycin, fetal calf serum (FCS), bovine serum albumin (BSA), and phosphate-buffered saline (PBS) from Eurobio (Les Ullis Cedex, France); sodium citrate dihydrate #3646-01 from J.T.Baker—Fisher Scientific Italia (Rodano, Italy); ECL System from Amersham Pharmacia (Buckinghamshire, UK), Bio-Rad assay and prestained Bio-Rad (München, Germany); protease inhibitor cocktail tablets from Roche Diagnostics (Meylan, France); 2’-7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (#D399), MitoSOX™ Red mitochondrial superoxide indicator (#M36008), MitoTracker Red CMXRos (#M7512), Lysostracker red DND-99 (#L7528), DRAQ5 (#62254), and Ly 294002 (#PHZ1144) from Thermo Fisher Scientific—Invitrogen, (Carlsbad, CA, USA).
4
Western Blot Analysis of Cytokine Signaling
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Recombinant human (rh) IL-6, IL-1α, TGF-β1, TNF-α, IL-8 and granulocyte macrophage colony-stimulating factor were purchased from R&D Systems (Abingdon, UK). rhIL-1β and gelatin solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium cell culture media, antibiotics, L-glutamine, sodium pyruvate and trypsin-EDTA were purchased from Invitrogen (Eugene, OR, USA). Fetal calf serum (FCS) was from Lonza (Levallois, France). Acrylamide, sodium dodecyl sulfate (SDS), Tris, and bovine serum albumin were purchased from Eurobio (Les Ulis, France). Anti-collagen type I antibody was purchased from Millipore (Billerica, MA, USA). Anti-STAT3 and p-STAT3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-IL6R antibody was purchased from Abcam (Cambridge, UK). Anti-HSC70 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal secondary immunoglobulins/HRP were purchased from Dako (Les Ulis, France). The Pierce™ BCA protein assay kit, LDS sample buffer, MOPS SDS running buffer, 4–12% Bis-Tris Gel, Pierce® ECL western blotting substrate, antioxidant and sample reducing agent were purchased from Thermo Fisher Scientific (Saint-Herblain, France). Tris/glycine migration buffer and Trans-blot® Turbo™ transfer pack were purchased from Bio-Rad (Hercules, CA, USA).
5
Fibrinolytic Microvesicle Activity Assay
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After three washes, washed MV pellets were re-suspended in 100 µl HEPES (10 mM, NaCl 140 mM, pH 7.40). Multi-well plates (Thermo Fisher Scientific, Illkirch, France) were coated with 150 µl assay buffer made of 50 mM phosphate, 80 mM NaCl, 2 g/L bovine serum albumin (Eurobio, Courtaboeuf, France) and 0.01% Tween. Fibrinolytic MV activity was assessed on 1/1 MV–plasminogen/CBS0065 samples (plasminogen: South Bend, USA; CBS0065: Stago, Asnières-sur-Seine, France). Microvesicle-free plasma (pellet supernatant) was used a negative control, and urokinase (Euromedica, Diegem, Belgium) was used with a range from 0.005 U to 0.5 U. Absorbance variations were measured for 18 h at 405 and 490 nm at 37 °C with a thermostated spectrophotometer (Versamax). Results are expressed in urokinase concentration.
6
Isolation of Primary Mouse Hepatocytes
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Primary mouse hepatocytes were isolated according to retrograde perfusion approach [23 (link)]. Briefly, anesthetized C57/BL6 mice were perfused through the inferior vena cava with Hepes buffer added with Liberase (2.3 mg/ml Roche®) and CaCl2 to start in vivo hepatocyte dissociation. The liver was then transferred to the petri dish with Leibovitz medium, and liver capsule was broken to release hepatocytes before filtration, washed with Hepes buffer, and transferred to Williams' E medium. Alive hepatocytes were isolated on 35% gradient percoll before seeding on a collagen-covered dish in Williams' E medium added with 2 mM glutamine, 10 IU/ml penicillin, 10 μg/ml streptomycin, 10% SVF, and 5 μg/ml insulin. After 6 h, attached hepatocytes were cultured in maintenance medium and Williams' E medium in which SVF was replaced by 1 mg/ml bovine serum albumin (Eurobio) and 0.1 μM dexamethasone (Sigma).
7
Western Blot Protein Analysis
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25 µg of protein were separated on 4–20% gradient polyacrylamide gel (Bio-Rad, Hercules, CA, USA) by the SDS-PAGE system and then transferred to a nitrocellulose membrane. These membranes were then blocked for 1 h at room temperature with 5% of bovine serum albumin (Eurobio, Montpellier, France) in Tris-buffered saline (10 mM Tris, pH = 8; 150 mM NaCl) and 0.05% Tween 20. Membranes were then incubated overnight at 4 °C with primary antibodies: α-SMA (A5228, dilution: 1/5000, Sigma, St. Louis, MO, USA), GAPDH (SAB2500541, 1/5000, Sigma, St. Louis, MO, USA). After 3 TBST washes of 5 min each, membranes were incubated in appropriate secondary antibodies (1/5000, Dako, Produktionsvej, Denmark) 1 h at room temperature. Immunocomplexes were revealed by a chemiluminescence detection system (GE Healthcare, Chicago, IL, USA). Proteins bands were scanned (ImageScanner III; GE Healthcare, Chicago, IL, USA) and analyzed.
8
Protein Expression Analysis in Colon Samples
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Colon samples were homogenized in a lysis buffer containing Hepes 20 mM, KCl 300 mM, MgCl2 3 mM, DTT 1 mM, EDTA 0.2 mM with 0.25% NP40, 1% of phosphatase inhibitor and 0,5% of protease inhibitor. The homogenates were centrifugated at 12,000 g for 15 min at 4 °C and the supernatant was collected. Protein concentration was determined with the Bradford method. 25 µg of protein were separated on 4–20% gradient polyacrylamide gel (Bio-Rad) by SDS-PAGE system and then transferred to a nitrocellulose membrane. For immunoblotting, membranes were blocked for 1 h at room temperature with 5% of bovine serum albumin (Eurobio, France) in Tris-buffered saline (10 mM Tris, pH = 8; 150 mM NaCl) and 0.05% Tween 20 (TBST). Membranes were then incubated overnight at 4 °C with primary antibodies: α-SMA (A5228, 1/5000, Sigma), MMP-9 (AB19016, 1/1000, Merck, Germany), GAPDH (SAB2500541, 1/5000, Sigma). After 3 TBST washes of 5 min each, membranes were incubated in appropriate secondary antibodies (1/5000, Dako, Denmark) 1 h at room temperature. Immunocomplexes were revealed by chemiluminescence detection system (GE Healthcare). Proteins bands were scanned (ImageScanner III; GE Healthcare) and analysed.
9
Immortalized Myoblast Cell Lines for LGMDR9
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Human wild-type (wt) AB1190 cells are immortalized myoblasts established from muscular biopsies of healthy individuals, and FKRP 17PV cells are human immortalized myoblasts established from human LGMDR9 muscular biopsies characterized by a compound heterozygous FKRP mutation (c.899T > C and c.798_819dup/p.Val300A). Both cell lines were obtained from the MyoLine immortalization platform of the Myology Institute [31 (link)]. KO-FKRP myoblasts were generated from AB1190 cells. Both cells were maintained in the growth medium, Skeletal Muscle Cell Growth Medium (Promocell, Heidelberg, Germany, C-23060) supplemented with 12.5% bovine serum albumin (Eurobio, Les Ulis, France, CVFSVF00-01) at 37 °C in 5% CO2. Cells were seeded on 6-well plates (2.9 × 105/well) or 96-well plates (9.6 × 104/well), respectively, for Western blot/Simple Western and On-Cell Western, pre-coated with 3 mg/mL of collagen I bovine (StemCell Technologies, Vancouver, BC, Canada, #04902). After 48 h, myoblasts were differentiated using Skeletal Muscle Cell Differentiation Medium (Promocell, C-23061). After 4 days of differentiation, myotubes were collected for different analyses.
10
Cuttlebone Formation in Sepia officinalis
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Sepia officinalis adult shells were collected on beaches in Normandy (France). The maximal length of the cuttlebones was 30 cm. S. officinalis eggs were collected in Luc-sur-Mer (Normandie, France) and kept in the lab in artificial sea water (salinity: 35 ppt -closed circulatory system), at 18°C. Eggs were opened and the embryos (whose stage is determined by morphological characteristics) were immerged in sea-water with 5% Ethanol, following recommendations of Buttler-Struben et al. (2018) and Winlow et al. (2018) (link) and according to directive 2010-63-UE and French decree 2013-118.
Cuttlebones were obtained by dissection. All soft tissues were removed and the cuttlebones were kept dried. Naef (1922 Naef ( , 1928a Naef ( , 1928b) ) has divided the embryonic period into stages, specified later by Lemaire (1970) and Boletzky et al. (2016) . We used the Lemaire and Boletzky et al. staging in this study. Stages from the beginning of matrix shell formation (stage 24), beginning of mineralisation (and first chamber, stage 25) to early final stage 30 (6 to 8 chambers) were analysed.
Crab chitin (Sigma Aldrich) and Bovine serum albumin (BSA, Eurobio) were used as references for the organic components. A marine non biogenic aragonite (botryoidal aragonite) was selected as a mineral reference.
Cuttlebones were obtained by dissection. All soft tissues were removed and the cuttlebones were kept dried. Naef (1922 Naef ( , 1928a Naef ( , 1928b) ) has divided the embryonic period into stages, specified later by Lemaire (1970) and Boletzky et al. (2016) . We used the Lemaire and Boletzky et al. staging in this study. Stages from the beginning of matrix shell formation (stage 24), beginning of mineralisation (and first chamber, stage 25) to early final stage 30 (6 to 8 chambers) were analysed.
Crab chitin (Sigma Aldrich) and Bovine serum albumin (BSA, Eurobio) were used as references for the organic components. A marine non biogenic aragonite (botryoidal aragonite) was selected as a mineral reference.
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