Recombinant mouse Cst6 and human CST6 (both from R&D Systems, Minneapolis, MN, USA) were used as a standard and as a negative control, respectively, in concentrations varying from 5 to 0.156 ng/ml. The wells of a 96-well plate were coated overnight at 4°C with polyclonal rabbit anti-human CST6 antibody (2 (link)), followed by incubation with 1% bovine serum albumin (ICN Biomedicals, Aurora, OH, USA) and 1% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min. Subsequently, standards, controls, and samples (undiluted up to 32× diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30 min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min. The above incubation steps were performed at 37°C and separated by repeated washing steps with PBS/0.05% Tween-20. Chromogenic substrate 1-step Ultra TMB (Thermo Fisher Scientific) was used as substrate for detection and the reaction was stopped by adding 4 M H2SO4. Each well was measured for mouse Cst6 at an absorbance of 450 nm with an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).
Bovine serum albumin (bsa)
Manufactured by ICN Biomedicals
Sourced in United States
Bovine serum albumin (BSA) is a commonly used protein in various laboratory applications. It is a purified protein derived from bovine (cattle) serum. BSA is a versatile and widely used reagent due to its ability to stabilize and protect other proteins, act as a blocking agent, and serve as a standard in protein quantification assays.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by R&D Systems (1 mentions)
Elisa microplate reader, by Bio-Rad (1 mentions)
Avidin biotinylated horseradish peroxidase complex, by Vector Laboratories (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Recombinant mouse cst6, by R&D Systems (1 mentions)
1 step ultra tmb, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cellquest pro software, by BD (1 mentions)
Yeast extract, by Dia-M (1 mentions)
Sorbitol, by Dia-M (1 mentions)
Potassium chloride (kcl), by Dia-M (1 mentions)
Ethanol, by Dia-M (1 mentions)
Potassium ferricyanide, by Merck Group (1 mentions)
Antrin r10, by Kyoritsu Seiyaku (1 mentions)
Mouse insulin enzyme linked immunosorbent assay kit, by Mercodia (1 mentions)
Mouse anti porcine cd3e sprd pe cy5, by Southern Biotech (1 mentions)
Cd4a fitc, by Southern Biotech (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cd8a pe, by Southern Biotech (1 mentions)
11 protocols using bovine serum albumin (bsa)
1
Quantification of Mouse and Human Cystatin E/M
2
Characterization of Mesenchymal Stem Cell Markers
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The expression of mesenchymal stem cell-associated surface markers at passage 3 was analyzed with flow cytometry, to characterize the immunophenotype. Approximately 1×106 cells were fixed with 3.7% paraformaldehyde (Sigma-Aldrich; Merck KGaA) for 10 min and then re-suspended in phosphate-buffered saline (PBS) solution (Welegene) containing 1% bovine serum albumin (BSA) (ICN Biomedicals), for 30 min to block the nonspecific antibody-binding sites. Next, the cells were incubated with specific antibodies against CD34, CD13, CD90, and CD146 at 4°C for 1 h, followed by incubation with fluorescent secondary antibodies at room temperature for 1 h. All antibodies were purchased from BD Biosciences. The percentages of CD13, CD90, and CD146-positive, as well as CD34-negative cells, were measured with a fluorescence-activated cell sorting (FACS) caliber flow cytometer (Becton Dickinson Immunocytometry Systems). The data were analyzed using the Cell Quest Pro software (BD Biosciences).
3
Biosensor Fabrication Protocol with Expanded Graphite
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Monopotassium phosphate, sodium hydroxide, sodium chloride, anhydrous acetic acid, d -glucose, glutaraldehyde (Mosreaktiv, Moscow, Russia); bovine serum albumin (fraction V, protease-free) (ICN Biomedicals Inc., Maumee, OH, USA), 2,6-dichlorophenolindophenol sodium salt, chitosan (low molecular weight), Nafion 117 (5% solution), PEDOT:PSS (1.3 wt % dispersion in H2O), bacteriological agar, potassium ferricyanide (Merck, Burlington, MA, USA); ethanol, sorbitol, yeast extract, and potassium chloride (Dia-M, Moscow, Russia) were used. Three-contact screen-printed carbon electrodes were purchased from Color Electronics (Moscow, Russia) and were used as electrodes in biosensors. The working electrode was 3 mm in diameter and was surrounded by a graphite auxiliary electrode and a silver reference electrode. Thermally expanded graphite (TEG) synthesized as described in [28 (link)] was used as a basis for the working electrodes of MFCs.
4
Islet Insulin Release Assay with COX Inhibitors
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Groups of 10 islets, control or pretreated islets, were transferred to vials containing Krebs–Ringer bicarbonate buffer supplemented with 10 mmol/L HEPES and 2 mg/mL bovine serum albumin (BSA; ICN Biomedicals Inc. Aurora, Ohio, USA; hereafter referred to as KRBH buffer). The KRBH buffer contained 1.67 mmol/L D-glucose during the first hour of incubation at 37°C (O2/CO2, 95:5). The medium was then removed and replaced by KRBH supplemented with 16.7 mmol/L glucose, and the islets were then incubated for a second hour. As mentioned above, some islets had been cultured with different COX inhibitors for the final 1–2 days of culture, and these substances were added also during the release experiments. That is, SC 560 (3 µmol/L), FR 122047 (5 µmol/L), rofecoxib (10 µmol/L), or indomethacin (10 µmol/l), all of which were dissolved in DMSO at a final concentration of 0.1% (vol/vol), was added to the release medium throughout the 2-h period. The islets were harvested, following retrieval of medium, and homogenized by sonication in 200 µL redistilled water. DNA and insulin contents were then measured as previously described (24 (link)).
5
Rabbit Embryo Genetic Modification
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Mature Dutch Belted rabbits were purchased from Kitayama Labes (Nagano, Japan).
Fertilized embryos were obtained from mature female rabbits that had been treated with 75
IU of follicle stimulating hormone (Antrin R10; Kyoritsu Seiyaku, Tokyo, Japan) twice
daily for 3 days, followed by 100 IU of human chorionic gonadotropin (hCG; Gonatropin;
Teikoku Zoki, Tokyo, Japan) 12 h later. The does were mated with fertile males immediately
after hCG treatment. Twenty hours after mating, fertilized embryos (zygotes) were flushed
from the oviducts using warmed HEPES-buffered RD medium [3 (link)] containing 4 mg/ml of bovine serum albumin (ICN Biomedicals, Irvine, CA,
USA). The pronuclei of zygotes were microinjected with 5 ng/µl of the
pX330 plasmid. After pronuclear injection of the plasmid, the embryos were cultured in RD
medium at 37°C in 6% CO2 and 5% O2 in air for 24 h and then
transferred into the oviducts of pseudopregnant Japanese White (JW) rabbits (Kitayama
Labes) that had been treated with 100 IU hCG and given manual vulval stimulation 24 h
before transfer. To confirm germline transmission, a heterozygous mutant female rabbit
that had a targeted allele of the tyrosinase gene, was mated with male JW rabbit. For
genotyping of the offspring, skin samples were collected 2-days after birth, and genomic
DNAs were extracted and sequenced.
Fertilized embryos were obtained from mature female rabbits that had been treated with 75
IU of follicle stimulating hormone (Antrin R10; Kyoritsu Seiyaku, Tokyo, Japan) twice
daily for 3 days, followed by 100 IU of human chorionic gonadotropin (hCG; Gonatropin;
Teikoku Zoki, Tokyo, Japan) 12 h later. The does were mated with fertile males immediately
after hCG treatment. Twenty hours after mating, fertilized embryos (zygotes) were flushed
from the oviducts using warmed HEPES-buffered RD medium [3 (link)] containing 4 mg/ml of bovine serum albumin (ICN Biomedicals, Irvine, CA,
USA). The pronuclei of zygotes were microinjected with 5 ng/µl of the
pX330 plasmid. After pronuclear injection of the plasmid, the embryos were cultured in RD
medium at 37°C in 6% CO2 and 5% O2 in air for 24 h and then
transferred into the oviducts of pseudopregnant Japanese White (JW) rabbits (Kitayama
Labes) that had been treated with 100 IU hCG and given manual vulval stimulation 24 h
before transfer. To confirm germline transmission, a heterozygous mutant female rabbit
that had a targeted allele of the tyrosinase gene, was mated with male JW rabbit. For
genotyping of the offspring, skin samples were collected 2-days after birth, and genomic
DNAs were extracted and sequenced.
6
Islet Insulin Secretion Assay
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After 3 to 4 days of culture, groups of 10 islets, from WT and from Mmp9 À/À mice, were transferred to vials containing Krebs-Ringer bicarbonate buffer supplemented with 10 mmol/L HEPES, 3 mg/mL bovine serum albumin (ICN Biomedicals, Santa Ana, CA), and 10 mmol/L glucosamine; hereafter referred to as KRBH buffer. The KRBH buffer contained 1.67 mmol/L D-glucose during the first hour of incubation at 37 C (oxygen/carbon dioxide, 95:5). The medium was then removed and replaced by KRBH supplemented with 16.7 mmol/L glucose and the islets were then incubated for a second hour. After this second incubation, the islets were harvested, following retrieval of the medium, and homogenized by sonication in 200 mL of redistilled water. A fraction of the homogenate was mixed with acid-ethanol [0.18 mol/L HCl in 95% (v/v) ethanol] from which insulin was extracted overnight. Thus, these measurements were performed after exposure to both low and high glucose in all islets. Insulin contents in incubation media and homogenates were determined by a mouse insulin enzyme-linked immunosorbent assay kit (Mercodia AB, Uppsala, Sweden).
7
Porcine Immune Cell Analysis
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Blood samples were collected by venepuncture on the morning (08.00 hours) of day 21 after an overnight fast, and were injected into two vacuum tubes containing sodium heparin. The vacuum tubes were immediately placed on ice until they arrived at the veterinary hospital for the examination of leucocytes and flow cytometry analysis, respectively (within 2 h). Leucocyte examinations (neutrophil, lymphocyte and monocyte counts) were done through an automatic blood analyser. Total peripheral blood lymphocytes were separated from heparinised peripheral blood by separation medium, and were then stained with mouse anti-porcine CD3e-SPRD (PE-Cy5; catalogue no. 4510-13), CD4a-FITC (catalogue no. 4515-02) and CD8a-PE (catalogue no. 4520-09), which were purchased from Southern Biotechnology Associates. PBS (1 £ ; Gibco) and 1•0 % bovine serum albumin (ICN Biomedicals) were used as diluent and washing buffer. Flow cytometry analysis was performed on a FACSCalibur flow cytometer (Becton Dickinson) and was repeated for the same sample and compared for repeatability.
8
Cell Culture Media and Reagents
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Phosphate buffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM), fetal calf serum (FCS), L-glutamine, antibiotics combination of streptomycin and penicillin, and Trypsin-EDTA were all purchased from Biological Industries (Beit Haemek, Israel). BSA and Tween-20 were purchased from ICN Biomedicals, Inc. (Aurora, OH, USA).
9
Luteal Tissue Oxygen Sensitivity
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Corpora lutea were cut into thin slices (thickness 180 μm) using a special microtome designed for processing unfixed tissue (Krumdick Tissue Slicer). Subsequently, tissue slices were preincubated for 2 h in M199 (Sigma-Aldrich, Saint Louise, MO, USA) supplemented with 0.1% BSA (ICN Biomedicals, Inc., Costa Mesa, CA, USA) and antibiotics (penicillin and streptomycin; Sigma-Aldrich). Then, the media was removed, slices were washed with warm PBS and fresh M199 with 0.1% BSA, and antibiotics were added into each well. The slices were incubated in (1) a high concentration of O2 (20% O2, 5% CO2, 75% N2) as a control and (2) reduced concentrations of O2 (10% or 3% O2, 5% CO2, 85% or 92% N2, respectively) in 37 °C for 6 and 24 h. After incubation, media samples were collected to determine progesterone concentrations. Luteal slices were snap-frozen and kept in −80 °C for further analysis of gene expression.
10
Aromatase Activity Modulation by Testosterone
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The effect of testosterone on E 2 production In order to determine activity of aromatase we performed in vitro experiment using precision-cut luteal slices obtained on days 12 and 14 of the estrous cycle/pregnancy. The slices were obtained as previously described (Przygrodzka et al. 2014 (link)) and incubated (one slice per well) in culture medium M-199 medium (Sigma-Aldrich) supplemented with: 0.1% BSA (ICN Biomedicals, Inc., Costa Mesa, CA, USA), antibiotics and anti-fungal additive (amphotericin B; Sigma-Aldrich) for 24 h at 37 8C in a humidified atmosphere containing 95% air and 5% CO 2 . Slices were pre-incubated for 1.5 h in culture medium only and then incubated for 24 h with testosterone (10 and 1 mM). Afterwards, incubation media were collected and stored for later analysis of E 2 concentration using the RIA method (see section 'Determination of E 2 , P 4 , and testosterone concentrations').
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