TGF-β1 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). A 10 μg/ml stock solution was prepared in sterile 4 mM HCl containing 0.1% human (P6140, Biowest, Nuaillé, France) or bovine serum albumin (A7906, Sigma-Aldrich Sweden AB). The stock solution was kept at −20 °C. When used, TGF-β1 was added to a final concentration of 5 ng/ml 24 h after cell seeding. Cell culture medium was collected (see below) after every 72 h of incubation and 1 ml fresh medium without or with 5 ng/ml TGF-β1 was added to each well.
A7906
Manufactured by Merck Group
Sourced in United States, Spain, Germany
A7906 is a laboratory instrument designed for performing spectrophotometric analysis. It measures the absorption or transmission of light by a sample at specific wavelengths, enabling quantitative analysis of various chemical and biological compounds.
Automatically generated - may contain errors
Lab products found in correlation
P1379, by Merck Group (5 mentions)
Lsm 880, by Zeiss (5 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
T8787, by Merck Group (4 mentions)
D9542, by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
T9284, by Merck Group (2 mentions)
Fluoroprobe 546, by Thermo Fisher Scientific (2 mentions)
Alexa 555, by Thermo Fisher Scientific (2 mentions)
Leica tcs sp5, by Leica Microsystems (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
H 3300 250, by Vector Laboratories (2 mentions)
Dmi6000 inverted microscope, by Leica camera (2 mentions)
Ba 1000, by Vector Laboratories (2 mentions)
Axiocam mrm camera, by Zeiss (2 mentions)
Leica confocal microscope, by Leica camera (2 mentions)
Hfh10, by Thermo Fisher Scientific (2 mentions)
Alex fluor plus 647, by Thermo Fisher Scientific (2 mentions)
72 protocols using a7906
1
TGF-β1 Supplementation in Cell Culture
2
Immunostaining of Embryonic Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryosections from embryos collected at E12.5 were prepared and processed for staining as in Charoy et al., 2012 (link). For some experiments, chick embryo sections and open-book spinal cords were blocked in 6% BSA (A7906, Sigma) and 0.5% Triton (T9284, Sigma) diluted in PBS for 5 hr at room temperature. Sections were incubated overnight at room temperature with anti-PlxnA1 antibody (gift from Y. Yoshida), anti-Robo3 antibody (1/100, R and D, AF3076); anti-L1CAM antibody (1:100, A439 Abcam 123990), anti-NgCAM antibody (1:50, 8D9, DSHB), anti-BEN (1:50, BEN, DSHB) or an anti-PC2 antibody (1:100, 3533, Abcam) in 1% BSA diluted in PBS. Alexa 488, Alexa 555 (1/500, Invitrogen) and Fluoroprobe 546 (1/400) were used as secondary antibodies.
3
Rad52 Binding to Radiolabeled DNA Oligo
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gel mobility shift assays were performed essentially as previously described40 (link). The 5’-end of Oligo211 (5′-GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT-3′, 48 nt) was 32P labelled using γ-32P-ATP (Perkin Elmer, NEG002A, 3000 Ci mmol−1) and T4 polynucleotide kinase (New England Biolabs, M0201S). The 32P-labelled Oligo211 (2 nM in DNA molecules) and Rad52 (0, 25, 50, 100, 150, or 200 nM) were mixed in 10 µL of binding buffer (32 mM Tris-HCl pH 7.8, 40 mM KCl, 0.8 mM DTT, and 0.08 mg mL−1 bovine serum albumin (BSA; Sigma, A7906, fraction V)) and incubated at 30 °C for 10 min. After addition of 2 µL of 5× loading buffer (20 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 50% glycerol, and 0.1% bromophenol blue (BPB)), the reaction mixture was applied to 10% non-denaturing PAGE (acrylamide: bis-acrylamide = 37.5:1) in 1× TAE buffer (40 mM Tris-acetate and 1 mM EDTA) at 100 V in an ice-cold tank. Gels were dried on grade DE81 ion exchange cellulose chromatography paper (Whatman, 3658-915). Radioactive signals were detected using a BAS2500 phosphorimager (Fujifilm) and quantified with Image Gauge Software version 3.4 (Fujifilm).
4
Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in polymer dishes as described previously5 (link). Cells were fixed for 20 min using 4% (by weight) formaldehyde solution (Sigma-Aldrich 252,549). Subsequently, cells were permeabilized with 0.5% by volume Tween 20 (Sigma-Aldrich P1379) in PBS for 20 min, and then blocked with 1% by weight bovine serum albumin (BSA, Sigma-Aldrich A7906), 22.5 mg/mL glycine in PBST (0.1% by volume Tween 20 in PBS) for 30 min. Cells were incubated with the primary antibodies diluted in 1% BSA in PBST at 4 °C overnight. After that, cells were incubated with the secondary antibodies in 1% BSA at room temperature for 1 h. Finally, the nuclei were stained with DAPI or Hoechst (Thermofisher Scientific R37606 or R37605) fluorescent dyes. The primary antibody used: Atf2 (abcam ab32019), Beta-Catenin (abcam ab19381), GEF-H1 (abcam ab155785), Lef1 (abcam ab137872), Oct4 (abcam ab19857), RNA pol II (abcam ab5408), Smad4 (CST 46,535).
5
Immunoassay for Selective Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used recombinant human p-selectin/CD62P (ADP3-050, R&D systems), recombinant human apolipoproteinA1 (3664-AP, R&D systems) or blood proteins harvested by centrifugation or three layered filter system to evaluate the immunoassay using color dye. To measure the limits of detection of the assay, we used serial diluted recombinant proteins. One ul of recombinant protein solution was applied to the filter system. Filter three was blocked with 1% bovine serum albumin (A7906, Sigma) in phosphate buffered saline for 10 minutes at room temperature. After brief washing with 0.1 M Tris buffer solution for 5 minutes, primary antibodies (anti-human SELP/P-selectin/CD62P antibody (LS-C296359, LSBio) and mouse anti-human ApoA1 antibody (MA5-14732, Invitrogen) were used for antigen-antibody reaction. HRP-linked secondary antibody (#.7074, Cell Signaling) was incubated for one hour at room temperature and followed by color reaction with 3,3´,5,5´-tetramethylbenzidine (TMB)-D solution (4600, Kem-En-Tec Diagnostics). When HRP conjugated mouse anti-human IgG antibody (SA1-35470, ThermoFisher Scientific) and HRP linked goat anti-human IgM antibody (31415, ThermoFisher Scientific) were used, color reaction with TMB solution was made directly. Enhanced chemiluminescence (ECL) using PicoEPD Western reagent (EBP1073, ELPIS-BIOTECH) was performed to compare the result with TMB solution.
6
Western Blotting Procedure for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as described previously (Hooper et al., 2022 (link)). Briefly, cell lysates were run on SDS-PAGE gels, transferred to PVDF membrane (Immobilon-P; Millipore), blocked with 5% BSA (A7906; Sigma-Aldrich)/TBS-T for 1 h, RT, and then incubated with primary antibody at 4°C overnight. Membranes were washed 3 × for 10 min in TBS-T and incubated with HRP-conjugated secondary antibodies (7074, 7076; Cell Signalling) for 45 min, RT. Membranes were washed again 3 × for 10 min in TBS-T and then developed with ECL (RPN2209; GE). Blots were scanned (V550; Epson Perfection).
7
Quinoa and Chia Seed Extracts in Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PPIs to be tested in cell cultures were obtained from seeds of C. quinoa and S. hispanica [6 (link)] obtained from local supermarkets. The protein concentration of the extracts (<30 KDa) was quantified in order to normalize the contents cell cultures were exposed to. Working solutions in EMEM were added to the cells to reach a final concentration of 100 µg·mL−1 (0.56% AU protease inhibitory activity) and incubated for 4 h. This concentration was established as effective at modulating immunonutritonal parameters in a preclinical model of severe liver dysfunction [4 (link)]. Control cells were used throughout exposed to either bovine serum albumin (heat shock fraction) (A7906, Sigma-Aldrich, Madrid, Spain), CM3 (A1520, Sigma-Aldrich) or bacterial lipopolysaccharide (LPS from E. coli) (L3129, Sigma-Aldrich). The assays were performed in two different days (n = 4).
8
Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on coverslips coated with poly-d -lysine (Sigma-Aldrich, P7280) and incubated in standard conditions. Cells on coverslips were fixed with ice-cold methanol at 4 °C for 30 min. Cells were washed with PBS multiple times and incubated with 5% BSA (Sigma-Aldrich, A7906) at room temperature for 30 min. Next coverslips were incubated with primary antibodies at the indicated concentrations against pericentrin (0.1 μg ml–1; Abcam, ab4448) and α-tubulin (0.5 μg ml–1; Sigma-Aldrich, T6074) diluted in 1% BSA for 1 h at room temperature in a humidified chamber. Coverslips were washed with PBS and incubated with the fluorescent secondary antibodies anti-mouse IgG-Alexa Fluor 594 (2 μg ml–1; Thermo Fisher Scientific, A-11005) and anti-rabbit IgG-Alexa Fluor 488 (2 μg ml–1; Thermo Fisher Scientific, A-11034) diluted in 1% BSA for 1 h at room temperature in the dark. Coverslips were washed in PBS followed by mounting and counterstaining with DAPI with ProLong Diamond antifade mountant. Cell images were captured at ×63 resolution on a Zeiss Axioplan upright microscope. Images were analysed using Fiji (v.2.9.0)53 (link).
9
Immunostaining of Neutrophil Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serially cut frozen slides were air-dried and fixed in prechilled 100% acetone). Slides were rinsed in distilled water and then washed in TBS-T. Endogenous peroxidase activity was quenched via incubation with 5% (v/v) H2O2 (Sigma-Aldrich, H3410) in dH2O. After 30 minutes, the slides were washed and incubated in serum-free protein block (DAKO, X0909) for 30 minutes, after which excess liquid was removed. Serial sections were then incubated with 1:1000 (v/v) anti-MPO (mouse, monoclonal; Abcam, ab25989) or 1:1000 (v/v) anti-NE (rabbit, monoclonal; Abcam, ab131260) or 1:500 (v/v) anti-CitH3 (rabbit, monoclonal; Abcam, ab219407) for 1 hour in antibody diluent (bovine serum albumin [1% w/v], Sigma-Aldrich, A7906) and Triton-X100 (0.5% v/v, Sigma-Aldrich, 270733) in TBS-T. Slides were further washed in TBS-T and incubated with horse radish peroxidase (HRP)-coupled secondary antibody (anti-mouse, DAKO, K4001; anti-rabbit, K4003) for 30 minutes. Following washing in TBS-T, slides were incubated for 10 minutes with Opal690 fluorophore (PerkinElmer, FP1497) in tyramide signal amplification (TSA) buffer (PerkinElmer, FP1498, 1:50 v/v dilution in 1x plus amplification buffer) and subsequently washed. Specimens were incubated in a 1:800 (v/v) dilution of Spectral DAPI for 10 minutes, washed, and then cover-slipped with fluorescence mounting medium.
10
Cytoskeleton and Nuclei Visualization in hSC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 5 and 10 days of hSC culture, samples were rinsed twice in PB 0.1M and fixed in 4% paraformaldehyde (PFA; 47608, Sigma-Aldrich, Madrid, Spain) for 20 min at room temperature. After cell fixation, PFA residues were removed with 3 washes of 10 min with DPBS. Then, cells were permeabilized and blocked in DPBS with 3% bovine serum albumin (BSA; A7906, Sigma-Aldrich, Madrid, Spain) and 0.1% Tween20 (P1379, Sigma-Aldrich, Madrid, Spain) for 1 h at room temperature.
To observe the cytoskeleton of the cells, samples were incubated with Alexa Fluor™ 555 Phalloidin (A34055, Thermo Fisher Scientific, Madrid, Spain) and diluted 1:200 at room temperature for 1 h. Cell nuclei were stained with DAPI (1/1000, D9542, Sigma-Aldrich, Madrid, Spain) for 10 min. Finally, the conduits were cut longitudinally to obtain a complete view of the lumina, and a confocal microscope (LEICA TCS SP5, Leica microsystems, Madrid, Spain) was used to obtain the images.
To observe the cytoskeleton of the cells, samples were incubated with Alexa Fluor™ 555 Phalloidin (A34055, Thermo Fisher Scientific, Madrid, Spain) and diluted 1:200 at room temperature for 1 h. Cell nuclei were stained with DAPI (1/1000, D9542, Sigma-Aldrich, Madrid, Spain) for 10 min. Finally, the conduits were cut longitudinally to obtain a complete view of the lumina, and a confocal microscope (LEICA TCS SP5, Leica microsystems, Madrid, Spain) was used to obtain the images.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!