Ground samples of ca. 100 mg were homogenized in 4 mL of deionized water and centrifuged at 4000 rpm for 5 min.; the supernatant (0.2 mL) was then added to 0.8 mL of Bradford solution (Sigma-Aldrich, St. Louis, MO, USA). Samples were read at 595 nm with a UV-Vis spectrophotometer (Agilent 8453; Santa Clara, CA, USA). Quantification was achieved using a calibration curve (10–100 µg/mL) with bovine albumin (Sigma-Aldrich), and the results were expressed as mg of bovine albumin equivalent on a fresh weight basis (mg BAE/g fw).
Bovine albumin
Manufactured by Merck Group
Sourced in United States, Germany, France, Poland, Italy, Brazil
Bovine albumin is a laboratory product derived from the blood serum of cattle. It is a common protein used in various cell culture and research applications to support cell growth and provide a nutrient-rich environment for cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated anti mouse antibody, by Cytiva (4 mentions)
Ecl kit, by Cytiva (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bradford assay, by Bio-Rad (4 mentions)
Lsm 510, by Zeiss (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Tgx precast gel, by Bio-Rad (3 mentions)
Geliance 600, by PerkinElmer (3 mentions)
Blue dextran, by Merck Group (3 mentions)
Pyrogallol, by Merck Group (3 mentions)
Folin ciocalteu reagent, by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Proteinase inhibitor cocktail, by Merck Group (2 mentions)
Pargyline, by Merck Group (2 mentions)
Collagenase, by Merck Group (2 mentions)
Pentobarbital sodium, by Merck Group (2 mentions)
Folin s reagent, by Merck Group (2 mentions)
95 protocols using bovine albumin
1
Protein Quantification Using Bradford Assay
2
Standardized Protein Quantification Protocol
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Protein concentrations of tissue extracts were determined by using Bio-Rad Protein Assay–Dye Reagent Concentrate, following the protocol of Bio-Rad Laboratories, Richmond, CA. Protein assays were done in order to express cytokine concentrations in the samples per milligram of protein. Stock protein standard solution of 1mg/ml was prepared by using bovine albumin purchased from Sigma Chemical Co., St. Louis, MO dissolved in distilled water. Blank and serial dilutions of standard (1 mg/ml, 0.8 mg/ml, 0.6 mg/ml, 0.4 mg/ml and 0.2 mg/ml) were used to generate the standard curve. The 15 μl aliquot of tissue extract was diluted in 30 μl distilled water (total volume 45 μl) and 10 μl of this dilution was used to load a single well of a Costar 96 well plate. All protein assays were done in triplicate. The dye reagent concentrate was diluted 1:4 with distilled water and 200 μl of this diluted dye was added to each of the wells. The plates were incubated on a shaker for 10 min and then absorbance was measured at 595 nm on a Clariostar plate reader (BMG Labtech, GmbH, Ottenberg, Germany) and analyzed using Mars Data Analysis Software (Version 3.01R2, 2013 BMG Labtech). A standard curve was plotted and used for calculating protein concentrations of tissue extracts. The protein concentrations were expressed as mg/ml.
3
Quantification of TLR3 and TLR7 Proteins
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TLR3 and TLR7 protein expression were quantified by Western blot assay. Whole cell lysates were treated with RIPA buffer containing proteinase inhibitor cocktail (Sigma-Aldrich, Merck Life Science S.r.l., Milan, Italy). Protein contents were evaluated by means of the Bradford assay (Bio-Rad, Milan, Italy) using bovine albumin (Sigma-Aldrich) as standard. Then, 20 µg of total proteins were loaded in each well and evaluated in denaturing conditions in 10% TGX pre-cast gel (Bio-Rad, Milan, Italy), with subsequent electroblotting transfer onto a PVDF membrane (Millipore, Merck Life Science S.r.l., Milan, Italy). The membrane was incubated with a specific antibody for the protein to be analyzed, then with a horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:5000; Amersham Biosciences, Piscataway, NJ, USA) and developed with the ECL kit (Amersham Biosciences, NJ, USA). The images were acquired by Geliance 600 (Perkin Elmer, Milan, Italy). The specific antibodies used were as follows: anti-TLR3 (clone 27N3D4), anti-TLR7 (clone NBP2-24905) (Novus Biologics, Milan, Italy). The complete Western blots are reported in Supplementary Figure S2 .
4
Spleen Cell Migration Assay
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Spleen cells were suspended in RPMI 1640 supplemented with 4 mg/ml fatty acid–free bovine albumin (Sigma-Aldrich). The same medium was used to prepare S1P (Sigma-Aldrich) at 10−8 M or chemokines at 50 ng/ml (R&D Systems). Cell migration was analyzed in Transwell chambers (Costar) with 5-µm-pore-width polycarbonate filters. Transmigrated cells were stained for CD3, NK1.1, CD27, and CD11b and counted by flow cytometry.
5
Radioactive Tracer Synthesis and Assay
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2-[1, 2-3H]-D-deoxyglucose, [7-14C]-benzylamine and the liquid scintillation cocktail Emulsifier-Safe were from Perkin Elmer SAS (Waltham, MA, USA). [14C]-tyramine was obtained from Sigma-Aldrich-Merck (Saint Quentin Fallavier, France). N-methyltyramine, p-tyramine, pargyline, synephrine, bovine albumin, collagenase and most of the other chemicals were from Sigma-Aldrich-Merck (Saint Quentin Fallavier, France). The indane hydrazino alcohol BTT 2052 was a generous gift from D. Smith and M. Salmi (Turku, Finland). The 2-benzofuranyl-2-imidazoline (BFI) was kindly given by Dr. A. Hudson (Univ. of Alberta, Edmonton, AB, Canada).
6
Radiolabeling of Murine TNF and Bovine Albumin
7
Quantifying α-Synuclein and Phosphorylation
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Cells were cultured onto glass-slides and fixed with 4% PFA for 20 min. Samples were blocked in PBS 1X containing 5% Normal Horse Serum and 5% bovine albumin (Sigma-Aldrich) for 1h. After washing in PBS 1X, cells were incubated with primary antibodies anti-αSyn (1:250, Abcam); anti-pser129 αSyn, (1:100; Abcam) for 2 h at room temperature, followed by PBS washes and incubation with secondary antibody (anti-mouse Alexa-488, 1:500; Thermo-Fischer) for 1 h at room temperature. Fluorescence intensity of αSyn and αSyn-p-serine-129 was quantified on at least 25 cells from 3 different experiments. Data are expressed as percentage relative to the untreated group (control). The images were obtained using a Leica SP8 confocal microscope, using a ×63 objective lens, 1.4 NA.
Total RNA was extracted using TRIzol Reagent (Life Technologies, Grand Island, NY) and cDNA generated with ThermoScript (Invitrogen). real-time PCR analysis based upon the intercalation of SYBR® Green on an ABI prism 7900 HT Fast Real Time PCR system (PE Applied Biosystems). The expression level of each gene was normalized to GAPDH. Samples were analyzed in triplicate from 5 different mouse ileum specimens.
Total RNA was extracted using TRIzol Reagent (Life Technologies, Grand Island, NY) and cDNA generated with ThermoScript (Invitrogen). real-time PCR analysis based upon the intercalation of SYBR® Green on an ABI prism 7900 HT Fast Real Time PCR system (PE Applied Biosystems). The expression level of each gene was normalized to GAPDH. Samples were analyzed in triplicate from 5 different mouse ileum specimens.
8
Immobilization of Biotin-Labeled DNA Origami
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For fluid exchange, a custom flow chamber was constructed similar as in cellular imaging. For binding of the origami structures to the surface of the flow chamber first, 20 µl of biotin-labelled bovine albumin (Sigma Aldrich, St. Louis, MO; 1 mg/ml, in buffer A) was applied to the chamber and incubated for 2 min. The chamber was then washed using 40 µl of buffer A. 20 µl of streptavidin (0.5 mg/ml, in buffer A) was then flown through the chamber and allowed to bind for 2 min. After washing with 40 µl of buffer A and subsequently with 40 µl of buffer B, 20 µl of biotin-labelled DNA structures (~300 pM) in buffer B were finally flown into the chamber and incubated for 5 min. The chamber was washed using 40 µl of buffer B.
9
Culturing Ovarian Cancer Cell Lines
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The OC cell lines SKOV-3 (RRID:CVCL_0532) and IGROV-1 (RRID:CVCL_1304) were cultured in Roswell Park Memorial Institute medium 1640 (RPMI; Gibco, #11875091) supplemented with 10% fetal calf serum (FCS; Integro), and OVCAR-3 (RRID:CVCL_0465) was cultured with RPMI supplemented with 20% FCS and 1 µg/mL bovine albumin (Sigma, #I0516). K562 (RRID:CVCL_0004) was cultured in Iscove’s Modified Dulbecco’s medium (IMDM; Gibco, #12440061) containing 10% FCS. Cell lines were tested for mycoplasma contamination with MycoAlertTM Mycoplasma Detection Kit (Lonza, #LT07-418) every 6 months. All cell lines were cultured for a maximum of 3 months. SKOV-3 and K562 were purchased from the ATCC. IGROV-1 and OVCAR-3 were a kind gift from Prof. Dr. OC Boerman, Department of Nuclear Medicine, Radboud University Medical Center, Nijmegen, the Netherlands. Primary low-grade serous OC cell line ASC009 was generated by NTRC in Oss, the Netherlands, from primary ascites material and a kind gift from Guido Zaman.
10
Histological Analysis of Ischemic Liver Tissue
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Tissue biopsies collected from the ischemic lobes were formalin fixed and paraffin embedded for hematoxylin-eosin staining (H&E). The severity of histological damage was blindly scored by an experienced liver pathologist (E.D.) using the Suzuki’s Score: according to this system, congestion, ballooning degeneration and necrosis are graded from 0 to 4 [27 (link)].
HLSC-EV uptake was analyzed by immunofluorescence microscopy. After rinsing in PBS, slices were incubated for 5 min at 4°C with a permeabilization solution containing 20 mmol/l Hepes, 50 mmol/L NaCL, 300 mmol/L sucrose, 3 mmol/L MagCl2, 0,5% Triton X- 100, pH 7.4. After washing with PBS, slices were incubated for 1 h at room temperature with a blocking solution of PBS added with 3% bovine albumin (both from Sigma-Aldrich) and incubated overnight at 4°C with an anti-mouse cytokeratin-8 primary antibody (1:200) (Abcam). At the end of the incubation, they were washed with PBS and then incubated for 1 h at room temperature with the Alexa Fluor 488-conjugated secondary antibody (1:200) (Invitrogen). Thereby, slices were washed with PBS and nuclei were stained with Hoechst. After a final washing in PBS, slides were mounted with Fluoromount (Sigma-Aldrich). Microscopy analysis was performed using a Cell Observer SD-ApoTome laser scanning system (Carl Zeiss).
HLSC-EV uptake was analyzed by immunofluorescence microscopy. After rinsing in PBS, slices were incubated for 5 min at 4°C with a permeabilization solution containing 20 mmol/l Hepes, 50 mmol/L NaCL, 300 mmol/L sucrose, 3 mmol/L MagCl2, 0,5% Triton X- 100, pH 7.4. After washing with PBS, slices were incubated for 1 h at room temperature with a blocking solution of PBS added with 3% bovine albumin (both from Sigma-Aldrich) and incubated overnight at 4°C with an anti-mouse cytokeratin-8 primary antibody (1:200) (Abcam). At the end of the incubation, they were washed with PBS and then incubated for 1 h at room temperature with the Alexa Fluor 488-conjugated secondary antibody (1:200) (Invitrogen). Thereby, slices were washed with PBS and nuclei were stained with Hoechst. After a final washing in PBS, slides were mounted with Fluoromount (Sigma-Aldrich). Microscopy analysis was performed using a Cell Observer SD-ApoTome laser scanning system (Carl Zeiss).
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