The cells grown on coverslips were fixed in 4% (v/v) paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature or overnight at 4°C. After rinsing in PBS, the fixed cells were permeabilized and nonspecific epitopes were blocked using 2% bovine serum albumin (Bovogen Biologicals, Keilor East, Vic, Australia) in 0.1% Tween-20/PBS, followed by incubation in the diluted primary antibody for 1 h at room temperature or overnight at 4°C. After three washes in PBS, samples were incubated for 1 h at room temperature with secondary antibodies diluted in PBS. Prepared samples were then mounted using Vectashield medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) and photographed using a fluorescence microscope (Nikon Corporation, Tokyo, Japan). The manufacturers and catalog numbers of the antibodies employed were as follows: mouse anti-myosin heavy chain (MHC; cat. no. MAB4470; R&D Systems) mouse anti-myogenin (cat. no. ab-1835; Abcam, Cambridge, MA, USA), mouse anti-desmin (cat. no. D1033; Sigma-Aldrich, St. Louis, MO, USA), Alexa-568 goat anti-mouse IgG (cat. no. A-11004), Alexa-488 goat anti-rabbit IgG (cat. no. A-11008) (both from Life Technologies). Quantification of immunofluorescence staining confirmed that four slides were used for each condition. Graphs represent the average of multiple tests from three independent experiments.
Bovine serum albumin (bsa)
Manufactured by Bovogen
Sourced in Australia, United States
Bovine serum albumin (BSA) is a protein derived from bovine blood serum. It is commonly used in various laboratory applications as a stabilizing agent, blocking reagent, and protein standard.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Fitc conjugated anti mouse igg antibody, by Merck Group (3 mentions)
Trtic conjugated anti rabbit igg antibody, by Merck Group (3 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse antibody, by Merck Group (3 mentions)
Nanosizer instrument, by Malvern Panalytical (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Ap color reagent, by Bio-Rad (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sunitinib malate su 11248, by Merck Group (2 mentions)
Pdcd4 d29c6 xp anti rabbit monoclonal antibody, by Cell Signaling Technology (2 mentions)
Amicon ultra lter, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Fluorogold, by Santa Cruz Biotechnology (2 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
44 protocols using bovine serum albumin (bsa)
1
Immunofluorescence Staining of Muscle Cells
2
Immunofluorescence Analysis of Germ Layer Markers
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To examine the expression of the three germ layer markers, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and blocked with 5% bovine serum albumin (BSA; BOVOGEN, Essendon, Australia) in 1X PBS-T (0.2% Triton X-100) for 1 hour at room temperature (RT). The primary antibodies were diluted in 1% BSA/1X PBS-T and were added to the samples for 1-2 hours at RT. The samples were then incubated with the secondary antibodies (either Alexa 488- or 594-conjugated antibody (Invitrogen)) for 40-60 min at RT. DAPI (10 μg/ml) (Sigma-Aldrich) was used to stain nuclei. The samples were analyzed with a Zeiss LSM510 confocal microscope (Carl Zeiss, Oberkochen, Germany). The antibodies used in this study are listed in Supplementary Table 2 .
3
Preparation of Mycobacterium tuberculosis H37Rv
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M. tuberculosis strain H37Rv was obtained from Nicholas P West (University of Queensland, Australia). Mycobacteria were grown statically to late-log phase at 37 °C in Middlebook 7H9 medium (BD Biosciences, San Jose, CA, USA) supplemented with 0.5% (v/v) glycerol (Ajax Finechem, Taren Point, Australia), 0.02% (v/v) Tween-80 (Sigma-Aldrich, St. Louis, MO, USA) and 10% (v/v) ADC supplements (50 g/l bovine serum albumin [Bovogen Biologicals, East Keilor, Australia], 8.5 g/l NaCl, 20 g/l dextrose and 0.03 g/l catalase [all from Sigma-Aldrich]), and 1 ml aliquots were stored at −80 °C until use. Four to five days prior to infection, mycobacterial stocks were thawed, passed 10 times through a 27-gauge needle to disperse aggregated bacteria, and cultured as above. On the day of infection, mycobacterial cultures were pelleted by centrifugation and resuspended in PBS + 0.05% Tween-80. To isolate single-celled Mtb, the suspension was then centrifuged at 130 g for 8 min to pellet aggregated mycobacteria. The supernatant (containing single-celled Mtb) was again pelleted and resuspended in either sterile water (for aerosolization) or PBS (for in vitro infections) to an optical density (590 nm) of 0.1–0.2. All procedures with viable Mtb were performed under biosafety level III conditions.
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M. tuberculosis strain H37Rv was obtained from Nicholas P West (University of Queensland, Australia). Mycobacteria were grown statically to late-log phase at 37 °C in Middlebook 7H9 medium (BD Biosciences, San Jose, CA, USA) supplemented with 0.5% (v/v) glycerol (Ajax Finechem, Taren Point, Australia), 0.02% (v/v) Tween-80 (Sigma-Aldrich, St. Louis, MO, USA) and 10% (v/v) ADC supplements (50 g/l bovine serum albumin [Bovogen Biologicals, East Keilor, Australia], 8.5 g/l NaCl, 20 g/l dextrose and 0.03 g/l catalase [all from Sigma-Aldrich]), and 1 ml aliquots were stored at −80 °C until use. Four to five days prior to infection, mycobacterial stocks were thawed, passed 10 times through a 27-gauge needle to disperse aggregated bacteria, and cultured as above. On the day of infection, mycobacterial cultures were pelleted by centrifugation and resuspended in PBS + 0.05% Tween-80. To isolate single-celled Mtb, the suspension was then centrifuged at 130 g for 8 min to pellet aggregated mycobacteria. The supernatant (containing single-celled Mtb) was again pelleted and resuspended in either sterile water (for aerosolization) or PBS (for in vitro infections) to an optical density (590 nm) of 0.1–0.2. All procedures with viable Mtb were performed under biosafety level III conditions.
4
Western Blot Analysis of Histone Acetylation
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Equal amounts of protein were fractionated on Bolt™ 4–12% Bis-Tris Plus Gels (ThermoFisher) following denaturing at 98 °C for 10 min. Proteins were transferred onto nitrocellulose membranes using the iBlot Gel Transfer Device (Invitrogen, Carlsbad, CA, USA) and blocked with 3% bovine serum albumin (Bovogen, Keilor East, VIC, Australia) in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. Membranes were incubated overnight with primary antibodies against acetylated H3K9 and acetylated H4 (S1, K5, K8, and K12) (Santa Cruz, Dallas, TX, USA) diluted in blocking buffer. GAPDH (Abcam) was used as a housekeeping reference protein. Membranes were incubated with goat anti-mouse IgG H&L horseradish peroxidase (HRP) (Abcam) in blocking buffer for at least one hour, then washed 3 times with TBST. Immunodetection was performed with the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) in the Chemi-Doc™ Gel Imaging System (Bio-Rad, Hercules, CA, USA). Band density was measured with Image Lab 6.0.1 software (Bio-Rad) and normalized to the density of housekeeping protein GAPDH or total protein as determined by Coomassie Blue staining.
5
IFN-γ ELISpot Assay for CD8+ and CD4+ T Cell Responses
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A 96 well MultiScreen enzyme-linked immunosorbent spot (ELISpot) plate (Millipore) was coated with anti-mouse IFN-γ antibody (clone AN-18, eBioscience, San Diego, CA, USA). The antibody-coated plate was blocked with 1% bovine serum albumin (Bovogen Biologicals, Essendon, Australia). Freshly-isolated splenocytes were dispensed into each well (500,000 cells/well), and 10 µg/mL of each single OLP was added. After 20 hours of incubation, the plate was washed with PBS and 0.05% PBST. Biotin-conjugated anti-mouse IFN-γ antibody (clone RA-6A2, eBioscience) and alkaline phosphatase-streptavidin (BD Pharmingen, San Diego, CA, USA) were sequentially added. A color reaction was then performed using AP color reagent (Bio-Rad, Hercules, CA, USA). The number of IFN-γ spot forming units was counted using an ELISpot reader (Cellular Technology Ltd., Cleveland, OH, USA). To evaluate CD8+ and CD4+ T cell responses separately, T cells were isolated from splenocytes using CD8 or CD4 microbeads (Miltenyi Biotec, Auburn, CA, USA), and IFN-γ ELISpot assays were performed with isolated T cells and T cell-depleted splenocytes [30 (link)].
6
Bone Marrow Isolation from Femurs
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To flush bone marrow, the femurs were first cleaned of connective tissue and muscle. Femurs were then washed with sterile phosphate-buffered saline (PBS). The bone was held in place with sterile tweezers, and the hip and knee joints were cut. The bone marrow was harvested from the proximal side of the right and left femurs. Bone marrow was flushed into a petri dish with a 26-gauge needle (Korea Vaccine, Ansan, Gyeonggi, Korea) attached to a 10-ml syringe filled with 0.5% bovine serum albumin (Bovogen, Melbourne, Australia) in PBS. The resulting cells were centrifuged at 3000 r.p.m. for 3 min at room temperature using a CytoSpin 4 Cytocentrifuge (Thermo, Waltham, MA, USA), then analyzed by immunostaining.
7
Mitochondrial Dynamics in A375SM Cells
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A375SM was seeded at a density of 3 × 103 cells in a circular coverslip pre-coated with gelatin in 24-well plates. After 24 h of stabilization, the cells were treated with various concentrations of the L-14 extract. Following 24 and 48 h of treatment, the culture medium was replaced with fresh medium containing 100 nM MitoTracker (Invitrogen, Carlsbad, CA, USA). The cells were incubated for 30 min at 37 °C and washed with PBS. IF staining was performed according to standard protocols. Briefly, washed cells on the coverslip were fixed with 4% paraformaldehyde, permeabilized with 0.15% Triton X-100 (Sigma), blocked with 3% bovine serum albumin (Bovogen, East Keilor, Australia), probed by anti-cytochrome c antibody (Novus Biologicals) and visualized using Alexa Fluor 647-conjugated secondary (Abcam). The coverslip was mounted with Hoechst 33,342 (Invitrogen). All samples were scanned via LSM 800 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) at 630× magnification.
8
Hepatic Lipogenesis and Fatty Acid Oxidation
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For all experiments, a modified Kreb's-Henseleit buffer was gassed for 40 min with 95% O2/5% CO2. Glucose (5 mM) and fatty acid-free BSA (4%) was added to the buffer immediately before experiments. All experiments were conducted in a shaking water bath at 30 °C.
For hepatic lipogenesis d-[3-3H]Glucose (TRK239; Amersham, Rydalmere, New South Wales, Australia) was added to the buffer to give a final concentration of 0.5 μCi/ml. Liver was sliced into 1–2 mm explants and incubated for 2 h, and the medium was removed. The tissue was washed in PBS and then homogenized in 1 ml PBS. The lipids were extracted in 2:1 chloroform-methanol, a 1-ml aliquot of the organic phase was removed, scintillation fluid was added, and radioactivity was counted in a liquid scintillation analyzer.
For analysis of oxidation all experiments, liver was sliced into 1–2 mm and explants were placed in warmed (30 °C) Krebs-Henseleit buffer pH 7.4 containing 2 mm pyruvate, 4% fatty acid-free bovine serum albumin (Bovogen, VIC, Australia) and 1 mm palmitic acid (Sigma, St Louis, MO, USA). After an initial incubation of 20 min, the incubation buffer was replaced with the same buffer described above supplemented with 0.5 μCi ml−1 of [1-14C]palmitate (Amersham BioSciences, Little Chalfont, UK).
For hepatic lipogenesis d-[3-3H]Glucose (TRK239; Amersham, Rydalmere, New South Wales, Australia) was added to the buffer to give a final concentration of 0.5 μCi/ml. Liver was sliced into 1–2 mm explants and incubated for 2 h, and the medium was removed. The tissue was washed in PBS and then homogenized in 1 ml PBS. The lipids were extracted in 2:1 chloroform-methanol, a 1-ml aliquot of the organic phase was removed, scintillation fluid was added, and radioactivity was counted in a liquid scintillation analyzer.
For analysis of oxidation all experiments, liver was sliced into 1–2 mm and explants were placed in warmed (30 °C) Krebs-Henseleit buffer pH 7.4 containing 2 mm pyruvate, 4% fatty acid-free bovine serum albumin (Bovogen, VIC, Australia) and 1 mm palmitic acid (Sigma, St Louis, MO, USA). After an initial incubation of 20 min, the incubation buffer was replaced with the same buffer described above supplemented with 0.5 μCi ml−1 of [1-14C]palmitate (Amersham BioSciences, Little Chalfont, UK).
9
Quantifying Mitochondrial ROS in A549 Cells
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Mitochondrial ROS was measured using MitoSOX Red with dihydroethidium conjugated to a mitochondria-localization tag. A549 cells were seeded at a density of 4×104 cells/well in a black 96-well plate, and the culture medium was changed the next day to 10% FBS-containing phenol red-free RPMI-1640 medium. Following genipin and elesclomol treatment, the culture medium was removed, and MitoSOX dye in HBSS buffer (Lonza Group, Ltd.) containing 2% bovine serum albumin (Bovogen Biologicals Pvt. Ltd.) was added. Following incubation at 37°C for 10 min, the buffer was changed to HBSS without dye, and fluorescence was measured on a Mithras LB 940 microplate reader at 510 nm excitation and 580 nm emission wavelengths.
10
Enzymatic Antioxidant Activity Evaluation
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Barium chloride dihydrate, ethanol, iron (II) sulfate heptahydrate, acetic acid, hydrochloric acid, nitric acid, ammonium sulfate, sodium hydroxide, phenol, and sulfuric acid were purchased from Daejung (Seoul, Korea). From Sigma-Aldrich (St. Louis, MO, USA), 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 2,2-azobis (2-methylpropionamidine) dihydrochloride, 1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid, glucose, Folin and Ciocalteu’s phenol reagent, peroxidase, α-(4-pyridyl N-oxide)-N-tert-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide, 2′,7′-dichlorofluorescein diacetate (DCF-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and gum arabic were purchased. RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin, and trypsin were purchased from Gibco (Mississauga, ON, Canada). Sodium carbonate anhydrous (Yakuri, Japan), bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Pittsburgh, PA, USA), bovine serum albumin (Bovogen, East Keilor VIC, Australia) and hydrogen peroxide (Junsei, Tokyo, Japan) were used. Commercial food-grade enzymes (alcalase (Al), α-chymotrypsin (α-chy), flavourzyme (Fla), kojizyme (Koj), neutrase (Neu), papain (Pap), pepsin (Pep), protamex (Pro), and trypsin (Try)) were purchased from Novozyme (Bagsvaerd, Copenhagen, Denmark).
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