DRG neurons and MSCs were washed with Phosphate-Buffered Saline (PBS; 0.1 M, pH 7.4) and fixed in 1% (v/v) paraformaldehyde (PFA, MM France, Brignais, France) for 10 min at room temperature (RT). After a wash with PBS, cells were permeabilized for 5 min with 0.1% (v/v) triton® X-100 (EDM Millipore, Billerica, MA, USA) and washed once more. Then, cells were blocked with 1% (w/v) bovine serum albumin (BSA, GE Healthcare, Chicago, IL, USA) for 30 min at RT to minimize the non-specific binding. Cells were further incubated with the primary antibody solution for 1 h at RT. After a wash with PBS, cells were incubated for 45 min in the dark with a solution containing the appropriated conjugated secondary antibody. Nuclei were counterstained for 5 min with 4′,6-diamidino-2-phenylindole (DAPI, 1:5000; Life Technologies™, Molecular Probes®). All dilutions were made in PBS. Images were acquired in a Leica TCS SPE Confocal Laser Scanning Microscope (Leica, Wetzlar, Germany).
Bovine serum albumin bsa
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Bovine serum albumin (BSA) is a well-known protein commonly used in various laboratory applications. It is a purified protein derived from bovine (cow) serum. BSA serves as a stabilizer, diluent, and blocking agent in numerous biochemical and cell culture procedures.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tcs spe confocal laser scanning microscope, by Leica (1 mentions)
Dylight 594 streptavidin, by Vector Laboratories (1 mentions)
Hoechst stain, by Thermo Fisher Scientific (1 mentions)
Poly d lysine hydrobromide, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
4 4 azobis 4 cyanovaleric acid acva, by Merck Group (1 mentions)
Methanol acs reagent, by Merck Group (1 mentions)
Trichloroacetic acid tca, by Merck Group (1 mentions)
Multiskan microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
U46619, by Merck Group (1 mentions)
P nitrophenyl phosphate pnpp, by Merck Group (1 mentions)
Citric acid, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Percoll, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
11 protocols using bovine serum albumin bsa
1
Immunocytochemistry of DRG Neurons and MSCs
2
Immunofluorescence Staining of EAAT2 in Cells
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Cells were grown on glass coverslips, which were previously treated with 0.1 mg/mL poly-d -lysine hydrobromide (Sigma-Aldrich). Cells were rinsed with phosphate buffered saline (PBS) and fixed in 4% (w/v) paraformaldehyde (PFA) solution (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at room temperature (RT), and then washed thrice with PBS. Non-specific binding sites were blocked with 0.5% (w/v) bovine serum albumin (BSA) (GE Healthcare, Chicago, IL, USA) solution for 30 min at RT. Cells were incubated with anti-EAAT2 antibodies (Millipore, Burlington, MA, USA) diluted 1:100 in 0.5% (w/v) BSA in PBS solution for 24 h at 4 °C. Cells were washed thrice with PBS at RT and incubated with a biotinylated horse anti-mouse IgG antibody, rat adsorbed (dilution 1:200; Vector Laboratories, Burlingame, CA, USA), for 1 h at RT, followed by a second incubation with a 1:100 dilution of avidin–biotin (Vector Laboratories) for 30 min. Once washed, the samples were incubated with DyLight 594 streptavidin (diluted 1:500 in PBS; Vector Laboratories) for 30 min at RT, followed by a last incubation step with Hoechst stain (diluted 1:6000; Invitrogen) for 5 min. Fluorescence was analyzed with a confocal microscope (LEICA AOBS-SP5X; Leica, Wetzlar, Germany).
3
Synthesis of Functional Polymer Nanoparticles
4
Quantification of Lipid Peroxidation by TBARS
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The malondialdehyde (MDA) content was quantitated as a marker of lipid peroxidation. This assay was based on the reaction between MDA and 2-thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS) to form a 1:2 MDA-TBA adduct. Hence, the amount of TBARS correlated with the MDA produced. To obtain the whole cell lysates, cells undergoing the same treatment as described above, were lysed in (radioimmunoprecipitation assay) RIPA buffer. The total protein concentration was determined by the DC protein assay (Biorad) using bovine serum albumin (BSA; GE Healthcare, Chicago, IL, USA) as the standard. 100 μl of whole cell extracts were then incubated with 100 μl of 10% (v/v) trichloroacetic acid (TCA; Sigma) and centrifuged. The resulting supernatants (100 μl) were mixed with 100 μl of 8% (v/v) SDS solution and 1 ml of 0.8% TBA solution in 10% acetic acid (Sigma) and boiled in 95 °C water bath for 1 hour. The mixtures were cooled and centrifuged. The absorbance was measured at 532 nm with the Multiskan microplate spectrophotometer (ThermoFisher Scientific). The concentration of TBARS was calculated using the MDA standard curve and is expressed as μM/mg of protein.
5
Platelet Activation Assay Protocol
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Prostaglandin E1 (PGE1), indomethacin, U46619, citric acid, trisodium citrate, ionomycin, glycerol, dextran-500, Nonidet P-40 (NP-40), fibrinogen, p-nitrophenyl phosphate (pNPP) and Percoll® were from Sigma (Poole, U.K.). Ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) was from Calbiochem (Nottingham, U.K.). BODIPY-FL-GDP, dithiothreitol, fluorescein (FITC)-conjugated anti-CD15 and allophycocyanin (APC)-conjugated anti-CD11b/CD18 antibodies were from Thermo Fisher Scientific, (Loughborough, U.K.). Alexa488-conjugated anti-mouse F(ab)2 was from Jackson ImmunoResearch (Ely, U.K.). Murine IgG2aκ isotype control antibody was from BioLegend (London, U.K.). (RS)-citalopram and PAF were from Cambridge Bioscience (Cambridge, U.K.). Horm® collagen was from Takeda (Linz, Austria). Bovine serum albumin (BSA) was from GE Healthcare (Buckinghamshire, U.K.). Fura-2 (AM) was from TEFLabs (Cambridge, U.K.). CRPXL was synthesised in the laboratory of Professor Richard Farndale (University of Cambridge, U.K.).
6
Isolation and Culture of Immune Cells
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BioWhittaker® Dulbecco's modified Eagle medium with glucose and l -glutamine (DMEM), Alpha minimum essential medium with desoxyribonucleotides, ribonucleotides and ultra-glutamine (α-MEM), RPMI-1640, Dulbecco's phosphate buffered saline, FCS and trypsin/EDTA with glucose were obtained from Lonza (Basel, Switzerland). Penicillin–streptomycin 100×, AB human serum, collagenase IV, ethylenediamine tetra-acetic acid (EDTA), Naphtol AS-MX, Fast Violet Salt B, Triton™ X-100, ssDNA from Salmon testes, Igepal® CA-630, alkaline buffer solution 1.5 M, phosphatase substrate, 4-nitrophenol, 7 μm polystyrene beads and Tween® 20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quant-iT™ PicoGreen® dsDNA Reagent was from Thermo Fisher Scientific (Waltham, MA, USA). Bovine serum albumin (BSA) was obtained from GE Healthcare (Chicago, IL, USA). Lymphoprep™ was bought from Stem Cell Technologies (Vancouver, Canada). Anti-mouse IgM microbeads, LS MACS™ columns and the QuadroMACS™ separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). All reagents were used as received and according to the manufacturer's recommendations.
7
Truncated Soluble Henipavirus Glycoprotein Expression
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The expression of a truncated soluble HeV G (sHeV G) protein has been described before [36 (link)], and the expression of a truncated soluble NiV G (sNiV G) protein in the L. tarentolae system (Jena Bioscience, Germany) was performed accordingly. Briefly, the NiV G sequence was codon optimized for the codon bias of L. tarentolae and the transmembrane domain and cytoplasmic tail were replaced by a double Strep-tag in order to enable affinity chromatography. The sequence product was cloned into the vector pLEXSY-sat2 (Jena Bioscience) using XbaI and NotI restriction sites to yield pLEXSYNiVG. The codon-optimized NiV G sequence was submitted to GenBank (accession No. MF379666). Transfection, clonal selection and subsequent protein purification were carried out as described previously [36 (link)]. Purity and size of the protein was evaluated by 10% SDS-PAGE and subsequent Coomassie blue staining. Protein concentration was determined by modified Bradford protein assay based on bovine serum albumin (BSA; GE Healthcare, Germany) as standard and according to the manufacturer’s instructions (Sigma Aldrich).
8
Fluorescent Labeling of Neuronal and Endothelial Cells
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SNs and ECs were fixed with 2% (w/v) paraformaldehyde for 30 min at room temperature (RT), permeabilised with 0.1% (v/v) Triton X-100 for 5 min at RT and blocked with 1% (w/v) bovine serum albumin (BSA, GE Healthcare) for 30 min at RT. For SNs, primary rabbit anti-rat β-III tubulin antibody (Abcam n°18207) was used at a 1:500 dilution at 4 °C overnight. Secondary goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen™ n°R37116) was used at a 1:400 dilution for 45 min at RT. Filamentous actin of ECs was labelled with rhodamin-phalloidin (Invitrogen™ n°R415) diluted at 1:400. Nuclei were labelled with DAPI (4′, 6′-diamidino-2-phenylindole, Life Technologies™) at 1 μg/mL for 5 min at room temperature. Images were acquired in a Leica TCS SPE 5 Confocal Laser Scanning Microscope.
9
Screening Cryptic Peptide Variants for uPA Cleavage
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To screen for uPA-cleavable cryptic PL3 peptide variants, we constructed a constrained phage library (configuration: AGRGRLVRXXXX, where X is a random amino acid). The phage library was cloned as described above, using partially randomized oligos (Table 1). Generated peptide-phage library pool was divided into two groups: non-treated and uPA pretreated. See below for uPA treatment protocol.
For cell-free phage display, 2 mg/mL of hexahistidinetagged recombinant b1 domain of NRP-1 (in-house) was coated onto Ni-NTA magnetic agarose beads (#36,113, Qiagen GmbH, Hilden, Germany) by incubating with endover-end mixing at RT for 1-1.5 h. Tris buffer (50 mM, pH 7.0) containing 5 mM imidazole, 1 M NaCl and 0.05% Igepal CA-630 (all from Thermo Scientific Inc.) was used for the dilutions; the same buffer with 0.1% bovine serum albumin (BSA; GE Healthcare, Little Chalfont, UK) was used for washes after the protein coating step. Proteincoated magnetic beads were incubated with phage library pools (5 × 10 9 pfu/mL) with end-over-end mixing for 1 h, followed by six washes with the washing buffer, and the release of protein-bound fraction with imidazole elution buffer (400 mM imidazole, 300 mM NaCl, 0.1% BSA and 0.05% Igepal CA-630 in PBS). Eluted phages were titered as described above.
For cell-free phage display, 2 mg/mL of hexahistidinetagged recombinant b1 domain of NRP-1 (in-house) was coated onto Ni-NTA magnetic agarose beads (#36,113, Qiagen GmbH, Hilden, Germany) by incubating with endover-end mixing at RT for 1-1.5 h. Tris buffer (50 mM, pH 7.0) containing 5 mM imidazole, 1 M NaCl and 0.05% Igepal CA-630 (all from Thermo Scientific Inc.) was used for the dilutions; the same buffer with 0.1% bovine serum albumin (BSA; GE Healthcare, Little Chalfont, UK) was used for washes after the protein coating step. Proteincoated magnetic beads were incubated with phage library pools (5 × 10 9 pfu/mL) with end-over-end mixing for 1 h, followed by six washes with the washing buffer, and the release of protein-bound fraction with imidazole elution buffer (400 mM imidazole, 300 mM NaCl, 0.1% BSA and 0.05% Igepal CA-630 in PBS). Eluted phages were titered as described above.
10
Immunofluorescence Staining of Cells
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For immunofluorescence staining, cells were washed twice with PBS and fixed with 3.7% formaldehyde (Merck, 1040031000) in PBS, followed by membrane permeabilization with 0.1% Triton-X-100 (Sigma, 93426) in PBS for 10 min at room temperature. Fixed cells were incubated with 3% Bovine serum albumin (BSA) (GE Healthcare Life Sciences) in PBS for 1 h followed by incubation with the primary antibody for 1 h in PBS with 1% BSA. After rinsing three times with PBS, staining was completed by Alexa Fluor®488-conjugated goat α rabbit antibody (Life Technologies, A11008) or Alexa Fluor®488-conjugated goat α mouse antibody (Life Technologies, A11001). Nuclei were visualized using DAPI nuclear counterstaining (Molecular Probes). Pictures of immunofluorescent cells were captured using an EVOS-fl fluorescence microscope (Advanced Microscopy Group) at 10 x magnification. Percentage of infected cells (relative to mock-treated) was calculated by counting infected cells in 10 x microscopic fields.
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