VL-17A cells were a generous gift of Dr. Dahn Clemens, University of Nebraska. Cells were maintained at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium (GE Healthcare Hyclone, Little Chalfont, UK) supplemented with 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 µg/ml). 1 × 106 cells were plated for RNA isolation, intracellular TG and non-esterified FA (NEFA) measurements. 3 × 106 cells were plated for all other assays. Cells were incubated with standard media; or supplemented with 100 mM ethanol and C2 ceramide (10 µM) (Cayman Chemical, Ann Arbor, MI), oleate (100 µM) (Sigma, St. Louis, MO) or palmitate (40 µM) (Sigma). oleate and palmitate were complexed to 5% FA free bovine serum albumin (BSA, Gemini Bioproducts, West Sacramento, CA) prior to addition to the media. Cells were given fresh media at 24 h intervals. Cell viability was assessed by CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI), according to manufacturer’s instructions.
Bovine serum albumin bsa
Manufactured by Gemini Bio
Sourced in United States
Bovine serum albumin (BSA) is a purified protein derived from bovine serum. It is commonly used as a stabilizing agent in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Promega (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Supersignal, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Sodium azide, by Thermo Fisher Scientific (2 mentions)
Fluorophore conjugated anti igg antibodies, by Biotium (2 mentions)
Oleate, by Merck Group (1 mentions)
Palmitate, by Merck Group (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Dulbecco s modified eagle s medium, by GE Healthcare (1 mentions)
Transporter 5, by Polysciences (1 mentions)
Fugene6, by Polysciences (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
A 21311, by Thermo Fisher Scientific (1 mentions)
Anti postsynaptic density protein 95 psd 95, by Cell Signaling Technology (1 mentions)
Anti glua1, by Cell Signaling Technology (1 mentions)
Anti fascin, by Abcam (1 mentions)
Anti synaptophysin, by Abcam (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Specific secondary antibody, by Thermo Fisher Scientific (1 mentions)
10 protocols using bovine serum albumin bsa
1
Lipid Metabolism Modulation in VL-17A Cells
2
Visualizing Golgi Protein Trafficking
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For capturing release of the RUSH reporters from the Golgi, HeLa cells on coverslips were transfected with U21-SBP-GFP or SBP-GFP-CDMPR (FuGENE6 or Transporter5, Polysciences, Warrington, PA; cat. no. 26008). RUSH reporters U21-SBP-GFP or SBP-GFP-CDMPR were released from the ER with biotin (40 µM) for 20 min at 37°C and then fixed in 4% paraformaldehyde. Cells were then washed with phosphate-buffered saline (PBS) and permeabilized with 0.5% saponin + 3% bovine serum albumin (BSA; Gemini BioProducts, West Sacramento, CA; cat. no. 700-100P) before being labeled with primary and then Alexa Fluor–conjugated secondary antibodies. All steps were performed in 0.5% saponin + 3% BSA, with the exception of three final PBS washes. For colocalization of GGA3 with U21-SBP-GFP or SBP-GFP-CDMPR, cells were instead fixed in methanol for 15 min at −20°C. For colocalization of clathrin with internalized α-U21 and α-class I MHC, Zenon-647 IgG1 (Invitrogen Z25008) was used to label α-clathrin heavy chain (X22).
3
Immunocytochemistry of Patterned Neurons
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Neurons were replated on 50-μm grids patterned on the bottom of 35-mm MatTek dishes (MatTek CatP35G-0-14-C) previously coated with 100 μg/ml PLO and 5 μg/ml laminin. Immediately after live imaging, cells were fixed in situ with 4% paraformaldehyde (PFA, Core Bio Services) for 30 min at room temperature. The cells were then washed three times in phosphate buffered saline (PBS), permeabilized with 0.2% Triton X-100 in PBS for 15 min and blocked with 2% bovine serum albumin (BSA, Gemini Bio-products) in PBS for 2 h at room temperature. Primary antibodies (chicken anti-MAP2, Abcam ab5392, 1:500; rabbit anti-GFP, Molecular Probes A-21311, 1:1,000) were diluted in blocking buffer (2% BSA in PBS). Cells were incubated with the primary antibodies overnight at 4°C. The following day cells were washed three times in PBS, and incubated with the secondary antibodies coupled to Alexa Fluor 488, 555, and 647 (Life Technologies, 1:1,000 in blocking buffer) for 1 h at room temperature. Cells were then washed three times in PBS to remove the secondary antibodies, and DAPI (1:10,000 in PBS) was added for 30 min at room temperature to counterstain cell nuclei. Cells were then washed again three times in PBS. Finally, the PBS was aspirated, and the cells were submerged in a drop of ProLong Gold anti-fade mountant (Life Technologies) and covered with a glass coverslip for subsequent analysis.
4
Western Blot Analysis of Synaptic Proteins
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Equal amounts of protein (20 μg) were separated on 10% Bis–Tris gel (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% (w/v) suspension of Bovine Serum Albumin (BSA; Gemini Bio-Products, West Sacramento, CA, USA) in 0.2% Tween 20 Tris-buffered saline (pH 7.5). After blocking, the membranes were incubated overnight at 4 °C with one of the following primary antibodies: anti-Drebrin (1:1000; Enzo Life Sciences), anti-GluA1 (1:1000; Cell Signaling), anti-p-GluA1 (Ser845; 1:1000; Cell Signaling), anti-postsynaptic density protein 95 (PSD95; 1:1000; Cell Signaling), anti-synaptic vesicle glycoprotein 2 (SV2A; 1:1000; Abcam), anti-fascin (1:1000; Abcam), anti-p-fascin (1:1000; Abcam), anti-synaptophysin (1:2000; Abcam), anti-β-tubulin (1:5000; Cell Signaling). The membranes were washed in tween-TBS for 20 min and incubated at 20 °C with the specific secondary antibody at a dilution of 1:10,000 (Pierce Biotechnology) for 60 min. The blots were developed using Super Signal (ThermoFisher Scientific, Rockford, IL, USA).
5
Western Blot Analysis of Synaptic Proteins
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Equal amounts of protein (30 μg) were separated on 10% Bis-Tris gel (Invitrogen, Carlsbad, CA), and transferred to nitrocellulose membranes that were blocked in a 5% (w/v) suspension of Bovine Serum Albumin (BSA; Gemini Bio-Products, West Sacramento, CA, USA) in 0.2% Tween 20 Tris-buffered saline (TBS-T, pH 7.5) for 1 h. Next, membranes were incubated overnight at 4 °C with the following primary antibodies: synaptophysin 1:1000 (Abcam, Cambridge, UK), PSD-95 1:1000 (Abcam, Cambridge, UK), profilin-1 1:1000 (Abcam, Cambridge, UK) and GAPDH 1:1000 (Santa Cruz Biotechnology, CA, USA). Membranes were then washed in Tween-TBS for 20 min and incubated with specific secondary antibodies at a dilution of 1:10,000 (Pierce Biotechnology) for 60 min. Immunocomplexes were visualized using Super Signal (ThermoFisher Scientific, Rockford, IL, USA) and band density measurements were made using ImageJ imaging software version 1.36b (NIH).
6
Immunostaining of 3D Bioprinted Constructs
7
Cytokine and Antibody Analysis Protocol
8
Immunofluorescence Analysis of VACV Infection
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Fluorescent microscopy for immunofluorescence was carried out using a Zeiss Axiostar plus epifluorescent microscope and images were captured with an Optronics camera system. Cells were grown on sterile glass coverslips, infected (MOI = 5) with ECTV, and fixed at the indicated time points with 10% formalin for 10 min at room temperature. For virus factory analysis, cells were then immediately mounted on a glass slide with ProLong Gold antifade reagent with 4’-6-diamidino-2-phenylindole (DAPI; Life Technologies) and allowed to cure overnight prior to visualization. For surface B5 staining, unpermeabilized cells were incubated—after fixation—in blocking buffer [1% bovine serum albumin (BSA; Gemini BioProducts) and 2% FBS (Gemini BioProducts) in 1x PBS] for 20 min and then incubated for 40 min with anti-B5 monoclonal antibody (BEI resources NR-553) diluted in blocking buffer. After three washes with 1x PBS, Alexa Fluor 568 goat anti-Mouse IgG1 (Life Technologies) diluted in blocking buffer was applied to cells for 20 min. Finally, the coverslips were mounted as described above on a glass slide with ProLong Gold antifade reagent with DAPI. A Leica DM-IL LED inverted epifluorescent microscope was used to visualize fluorescence from VACVΔE3LΔK3L infected cells. These images were then processed using QImaging software.
9
Immunostaining of Cellular Adhesion Proteins
10
Immunofluorescence Staining of Cardiac Cells
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