Different brain structures (HT, HIP, OB, RET, CX, and CB) were collected from mice (n = 7–10 mice) of 5–7 days of age immediately after sacrifice. Tissues were collected in D-PBS and were cut into small pieces of 1 mm3. Afterwards, tissues were digested using papain digestion solution containing 20 U/mL papain (ref LS003126, Worthington Biochemical Corporation, Lakewood, NJ, USA) and 200 U/mL DNase (ref D4527, Sigma Aldrich), for 1 hour at 37°C with constant rotation. This was followed by mechanical dissociation of the pellet for 3 times using a 0.02% (w/v) BSA solution (ref A4161, Sigma Aldrich) containing 333 U/mL DNase (ref D4527, Sigma Aldrich). This was followed by cell counting using a hemocytometer and centrifugation at 1000 rpm for 10 minutes. Cells were resuspended in a glia culture medium at 1000 cells/μL and plated at 200,000 cells/glass slide. Culture medium was DMEM medium (ref 41965–039, Gibco Life technologies,) supplemented with 10% fetal bovine serum (ref 16000–044, FBS-Invitrogen/Gibco), 2 mM glutamine (Sigma, G7513), 1 mM sodium pyruvate (ref 11360–039, Invitrogen/Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/Strep solution, ref 151 40–122, Invitrogen/Gibco). Cells were incubated at 37°C with 5% CO2 for 8 days and medium was changed every 3 days.
Bsa solution
Manufactured by Merck Group
Sourced in United States
BSA (Bovine Serum Albumin) solution is a laboratory reagent used as a protein standard and blocking agent in various biochemical and immunological assays. It is a purified form of the albumin protein derived from bovine serum. BSA solution is a clear, colorless liquid that is commonly used to quantify the concentration of other proteins in a sample and to prevent non-specific binding in assays.
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Lab products found in correlation
Imagequant las 4000 system, by GE Healthcare (2 mentions)
Mini protean tgx stain free gel 4 15, by Bio-Rad (2 mentions)
Iblot 2 pvdf regular stacks, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Glucose solution, by Merck Group (2 mentions)
Versafluor fluorometer, by Bio-Rad (2 mentions)
Multi wavelength absorbance detector, by Wyatt Technology (2 mentions)
1260 infinity 2 hplc, by Agilent Technologies (2 mentions)
Optilab rex differential refractive index, by Wyatt Technology (2 mentions)
Minidawn treos multi angle light scattering, by Wyatt Technology (2 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Pen strep solution, by Thermo Fisher Scientific (1 mentions)
Dnase, by Merck Group (1 mentions)
Papain, by Worthington (1 mentions)
Ab49900, by Abcam (1 mentions)
Paraformaldehyde solution, by Merck Group (1 mentions)
Triton x, by Merck Group (1 mentions)
Lab tek 2 chambered coverglass, by Thermo Fisher Scientific (1 mentions)
28 protocols using bsa solution
1
Primary Mouse Brain Cell Culture
2
Protein Expression Analysis Protocol
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Freshly harvested cells were lysed in RIPA buffer. Protein concentrations were determined by Pierce BCA™ Protein Assay Kit (Pierce). Proteins were separated via Mini Protean TGX stain free gel 4–15% (BioRad) and transferred to polyvinylidene difluoride membrane by using iBlot 2 PVDF Regular Stacks (Invitrogen) and iBlot system transfer system (LifeTechnologies). Membranes were blocked in 5% BSA solution (Sigma). Primary antibodies were diluted following the manufacturer’s instructions: anti-beta Actin, [AC15] (HRP-conjugated) ab 49900, Abcam (1:25000) and antiPTGR1 [EPR13451–10], ab181131, Abcam (1:1000). Signals were developed using Clarity Western ECL Substrate (BioRad) and Image Quant LAS4000 System (GEHealthCare).
3
Immunofluorescent Staining of BMDMs
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BMDMs were passed onto an OPN-coated 8-well Lab-Tek II chambered cover glass (Nalge Nunc International) at 1.5 × 104/well density. After incubation as described above, BMDMs were washed with chilled PBS buffer and fixed with 2% paraformaldehyde solution for 10 minutes at room temperature and permeabilized with 0.05% Triton-X (Sigma-Aldrich). Cells were blocked with 2% BSA solution (Sigma-Aldrich) and subjected to p16 and F4/80 antibody treatment overnight. Corresponding secondary antibodies conjugated with fluorescent dyes were used to visualize signal with DAPI counterstaining. Images were acquired as described above.
4
Quantifying Viral Neuraminidase Expression
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The MDCK cells were maintained on sterile glass coverslips and then infected with H1N1 A/PR/8/34 virus for 2 h. After that, the cells were transferred to new media containing the test compounds at various concentrations. The cultures were incubated at 37 °C under a 5% CO2 atmosphere for 24 h. After fixation and permeabilization, the cells were blocked with 1% bovine serum albumin (BSA) solution (Sigma, St. Louis, MO, USA) for 1 h and then incubated overnight with monoclonal neuraminidase antibody (Gene Tex) that was diluted 1:50 in PBS. The cells were then incubated with the secondary antibody FITC (fluorescence isothiocyanate)-conjugated goat anti-Rb IgG (Abcam, Cambridge, UK) for 1 h, and the nuclei were stained with 500 nM 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, USA) solution for 5 min at room temperature. The slides were mounted with mounting medium for fluorescence (Vectashield, Vector Lab, Inc., USA). The immunofluorescence images were acquired with fluorescence microscopy (Olympus ix70 Fluorescence Microscope, Olympus Corporation, Tokyo, Japan).
5
Evaluating TGF-β2-Induced TM Cell Viability
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Human TM cells between passages 5 and 7 were grown to 80% confluence and growth arrested using serum free medium prior to stimulation. Cells were stimulated with recombinant human TGF-β2 (R&D Systems, UK) at a concentration of 5 ng/mL for 24 h. Vehicle control cells were stimulated with equal volumes of 4 mM HCl and 0.1% BSA solution (Sigma, UK). The viability of TM cells treated with TGF-β2 was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT, Sigma-Aldrich, USA) (Supplemental Fig. 1 C). 24 h post TGF-β2 treatment, 10uM of MTT solution was added to each well and incubated for 3 h. After incubation, the media was removed and the formazan crystals were dissolved in 100 µl of DMSO. The optical densities (OD) of the dissolved formazan crystals was read on a plate reader at 570 nm (Omega FluroStar; US). The quantification of cell viability was obtained by comparing the optical density of the treated and untreated samples. The relative cell viability was calculated for each tissue as Arbitral Unit (AU), extrapolated by Optical Density (OD) of the samples.
6
Preparation of C-II Mimetic Peptides and Lipid Vesicles
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C-II-a and C-II-i mimetic peptides51 (link) were dissolved in PBS to a concentration of 2 mg/ml; VLDL (2.1 mg/ml) and LDL (2.9 mg/ml) were from healthy donors and isolated using a standard ultracentrifugation protocol in UC San Diego lipid core. DHA:BSA were made by dissolving 12 mg DHA (Cayman, Cat. 90310), which was first dried under argon, in 4 ml of 100 mg/ml BSA solution (Sigma, Cat. A8806). Oleic acid:BSA was from Sigma (Cat. O3008). Fatty acid concentrations were measured using an HR Series NEFA-HR method (Wako, Cat. 999–34691, 991–34891, 993–35191) and BSA was measured using Lowry assay (Biorad, Cat. 500–0116). To make POPC:TG-DHA and POPC liposomes, chloroform solubilized POPC (Avanti, Cat. 850457) and tridocosahexaenoin (TG-DHA) (Larodan Cat. 33–2260) were mixed at 2 mg POPC with 1.2 mg TG-DHA or 2 mg POPC alone. The mixtures were dried under argon in round bottom glass tubes and then 1 ml PBS was added and incubated at RT for 30 min. Then, POPC:TG-DHA or POPC in PBS was sonicated in a water bath ultrasound machine (VWR, Model 75D) three times for 10 min, until the solution became uniform and translucent.
7
SARS-CoV-2 Spike Protein Localization
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Cells were prepared onto salinized glass slides, fixed, and stained as previously described in the FISH process. Briefly, after fixation with 4% PFA, cells were washed with 0.1 M PBST pH 7.4. After, cells were incubated for 10 min with 0.1 M glycine and treated with BSA solution (Sigma) for 30 min. Cells were then incubated overnight at 4 °C with primary antibodies at 1:100 dilution in PBST and 1% BSA [SARS-CoV-2 Spike S1 antibody (#HC2001 GenScript—#A02038), p62 antibody (#BD 610832), Lamp1 antibody (#BD 555798) and CD63 antibody (#BD 556019), according to the desired double IF. The slides were washed and incubated for 2 h with secondary antibodies (Alexa 488 anti-Human IgG Thermo Fisher—#A11013 and Alexa Fluor 555 Anti-Mouse IgG #A21422), diluted 1:500 in PBST + 1% BSA. Cells were then washed and stained with DAPI (Santa Cruz Biotechnology, #SC3598). Subsequently, cells were washed with PBS, and the labels were mounted in an aqueous mounting solution for confocal imaging.
8
Anti-glycation Activity of AgNPs
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The anti-glycation activity of the AgNPs was measured as % inhibition of AGEs by following the protocol of Shah et al.19 The AgNP solution was mixed with 20 mg mL−1 BSA solution (Sigma Aldrich) prepared in phosphate buffer (0.1 M; pH 7.4), 1 mL of phosphate buffer (0.1 M; pH 7.4) containing sodium azide 0.02% (w/v) and glucose solution (0.5 M; Sigma Aldrich) prepared in phosphate buffer and incubated for 5 days at 37 °C in the dark. The formation of the fluorescent AGEs was verified using a VersaFluor fluorometer (Bio-Rad, France) with the emission and excitation wavelengths set at 410 nm and 330 nm, respectively.
9
Efficient Embryonic Stem Cell to Epiblast-like Cell Conversion
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EpiLCs induction was modified from47 (link). Briefly, ES cells were gently dissociated using trypsin/EDTA, and a single-cell suspension was plated onto Geltrex (GIBCO_#A1413202)-coated plates at a density of 5000 cells/cm2 in ESC growth medium. One day after plating, EpiLC induction started by adding the N2B27 medium supplemented with 20 ng/ml ActivinA (GIBCO_#PHC9564) and 12 ng/ml bFGF (GIBCO_#PHG0026). The cells were splitted 1:3 in small clumps using 1 mg/ml Collagenase IV (GIBCO_#17104019). Medium was changed daily. EpiLCs were collected for DNA, RNA and protein analyses after 14 days of induction.
N2B27 medium is composed by 50% advanced DMEM/F12 (GIBCO_#12634028) and 50% Neurobasal medium (GIBCO_#21103049), supplemented with 0.5% N2 Supplement (GIBCO_#17502048), 1% B27 Supplement (GIBCO_#17504044), 0.033% BSA solution (SIGMA_#A9647), 50 µM β-mercaptoethanol (Sigma_#M3148), 2 mM Glutamax (GIBCO_#35050038), 25 U/ml penicillin and 25 μg/ml streptomycin (Invitrogen_#15070063).
N2B27 medium is composed by 50% advanced DMEM/F12 (GIBCO_#12634028) and 50% Neurobasal medium (GIBCO_#21103049), supplemented with 0.5% N2 Supplement (GIBCO_#17502048), 1% B27 Supplement (GIBCO_#17504044), 0.033% BSA solution (SIGMA_#A9647), 50 µM β-mercaptoethanol (Sigma_#M3148), 2 mM Glutamax (GIBCO_#35050038), 25 U/ml penicillin and 25 μg/ml streptomycin (Invitrogen_#15070063).
10
Multiparametric Analysis of Cellular Phenotypes
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For antibody staining, cells were fixed with 2% Histofix (Roth) for 30 min and permeabilized by 0.05% Triton X-100 (Thermo) for 15 min. After blocking in 3% BSA solution (Sigma) for a minimum of one hour, cells were incubated with antibody in a 1% BSA solution (Supplementary Table S2 ). For autophagy, trypsinized cells were stained with CYTO-ID according to the protocol of the CYTO-ID® Autophagy detection kit (Enzo) using 5% FCS. CYTO-ID staining was for 45 min. For apoptosis, 5 mM or 10 mM H2O2 (AppliChem) was incubated with the cells (24 well) in cell culture medium. Incubation time was as described in the figure legend. Cells were then trypsinized and centrifuged (400g, 5 min) including the respective cell culture supernatants. The cell pellet was resuspended in 50 µl of the Annexin V Apoptosis Detection Kit/7-AAD solution (BioLegend) and incubated for 15 min. Flow cytometry analysis was accomplished in a CytoFlex Flow Cytometer (Beckman Coulter).
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