Whole cells lysates were prepared by pelleting 1 ml of an overnight culture diluted to an optical density at 600nm (OD600) of 1.0, and resuspending in 50 μl of distilled water plus 50 μl of 2x SDS loading buffer. SDS PAGE and transfer of proteins to a PVDF membrane for western blot were performed as previously described71 (link). Monospecific antisera against H1, H4 and H7 flagellin was purchased from the Statens Serum Institute, Denmark. Polyclonal antibodies against FimA (targeting the peptide CAGSVDQTVQLGQVRT) and LafA were generated by the Antibody Facility at the Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia). OmpA antiserum was purchased from the Antibody Research Corporation, USA (item #111120). Primary antibodies were detected with commercially purchased alkaline phosphatase-conjugated anti-rabbit antibody (Sigma Aldrich). SIGMAFASTTMBCIP®/NBT (Sigma Aldrich) were used as substrate for detection.
Sigmafast bcip nbt
Manufactured by Merck Group
Sourced in United States, United Kingdom
SigmaFast BCIP/NBT is a substrate system used in colorimetric detection of alkaline phosphatase-conjugated molecules in various immunoassay applications. It provides a reliable and consistent method for visualization of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin alkaline phosphatase, by Mabtech (8 mentions)
Elispot reader, by Cellular Technology (4 mentions)
Msips4510, by Merck Group (4 mentions)
Elispot plate, by Merck Group (4 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Extravidin alkaline phosphatase, by Merck Group (3 mentions)
Ks elispot reader, by Zeiss (3 mentions)
Extravidin ap, by Merck Group (3 mentions)
Mt307 biotin, by Mabtech (3 mentions)
Oil red o, by Merck Group (3 mentions)
Alkaline phosphatase conjugated anti rabbit antibody, by Merck Group (2 mentions)
Alcian blue, by Merck Group (2 mentions)
Monoclonal anti bovine ifnγ antibody, by Bio-Rad (2 mentions)
Primary rabbit polyclonal anti bovine ifnγ antibody, by Merck Group (2 mentions)
Anti mouse ifn γ capture antibody, by BD (2 mentions)
Ifn γ detection antibody, by BD (2 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris midi protein gels, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Akp streptavidin, by BD (2 mentions)
109 protocols using sigmafast bcip nbt
1
Whole Cell Lysate Preparation and Western Blot Analysis
2
Dermal Papilla Cell Response to Fucose and Chondroitin Disaccharide
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Dermal papilla cells were seeded at a density of 10,000 cells in each 8 Cell Culture Chamber FlexiPERM® (Sarstedt AG & Co., Nümbrecht, Germany) on a microscope slide. Cells were allowed to grow for 24 h and were incubated with appropriate concentrations of L-fucose and chondroitin disaccharide Δdi-4S sodium salt for 120 h. A BCIP/NBT tablet (SigmaFastTM BCIP-NBT; Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in 10 mL distilled water to prepare the substrate solution. Cells were washed with Wash Buffer (Tween20 in PBS) without disrupting the cell monolayer. After liquid aspiration, cells were covered to a depth of 2–3 mm with ice-cold 100% methanol and were allowed to fix for 15 min at −20 °C. Cells were then washed with Wash Buffer. The BCIP/NBT substrate solution was added to cover the cellular monolayer. The slides were incubated at room temperature in the dark for 10 min. The substrate solution was aspirated and the cell monolayer was washed with Wash Buffer. Cells were analyzed under a light microscope.
3
Ganglioside Extraction and Analysis
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All organic solvents were of LC-MS2 grade. DEAE sephadex A-25, was obtained from Pharmacia Biotech AB, Uppsala, Sweden. Preparative C18 125°A 55–105 µm, was obtained from Waters GmbH, Eschborn, Germany. Gangliosides GM3, GM2, GD2, GD3, and GD1a were obtained from Matreya, USA. Isotopically labelled D5-GM3 and D5-GM1 were purchased from Avanti Polar Lipids, and isotopically labelled D3-GM2 and D3-GD3 from Cayman Chemicals. GM1a was derived from FIDIA, Italy and Cronassial (bovine brain gangliosides containing mainly GM1a, GD1a, GD1b, and GT1b) from the company Dr. Madaus & Co, Cologne, Germany. HPTLC silica gel 60 F254 glass plates, Merck KGaA (Darmstadt, Germany). Plexigum (Poly(Isobutyl methacrylate)), SigmaAldrich (St. Louis, Missouri, USA). Mouse anti-human GD2 IgG2a antibody, clone 14. G2a, cat. #554272, BD Pharmingen (Franklin Lakes, New Jersey, USA). Alkaline phosphatase-conjugated goat anti-mouse IgG+IgM (H+L), cat. #115-055-068, Jackson ImmunoResearch Laboratories Inc. ((Farmington, CA, USA). SIGMAFASTTM BCIP®/NBT (5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium) tablets, Sigma Aldrich (St. Louis, Missouri, USA).
4
Alkaline Phosphatase Activity Assay
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Undifferentiated C3H10T1/2 cells show weak alkaline phosphatase (ALP) activity whereas differentiated osteoblasts show very high activity of ALP. ALP activity is therefore a good indicator of osteoblast differentiation. One BCIP/NBT tablet (SigmaFastTM BCIP-NBT, Sigma Aldrich) was dissolved in 10 ml distilled ddH20 to prepare the substrate solution, which detects ALP by staining the cells blue-violet when ALP is present. It should be stored in the dark and used within 2 h. The washing buffer was made by adding 0.05% Tween 20 to PBS without Ca++/Mg++ (Corning, #21-040-CM). Cells were washed one time with PBS and fixed with 4% paraformaldehyde for 60 s. After the fixation, cells were washed with washing buffer and then treated with BCIP/NBT substrate solution at room temperature in the dark for 10 min. The staining process is being observed every 2–3 min. Cells are then washed with 1× with washing buffer and 1× with PBS, respectively. After the last wash, the staining results were analyzed at the dissection microscope.
5
Quantifying Mineralization in Osteogenic Cultures
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hBM‐MSCs samples in hBM‐MSCs OM were analyzed at day 14 and SaOS‐2 cell samples in SaOS‐2 OM were analyzed at day 7. The cells were washed with PBS and fixed in 4% paraformaldehyde for 1 min. Cells were then washed once with PBS containing 0.05% Tween and stained with BCIP/NBT assay solution (SigmaFastTM BCIP‐NBT; Sigma Aldrich) for 10 min. The plates were washed three times with PBS with 0.05% Tween and air‐dried prior to scanning on a flatbed scanner. The percentage area of mineralization per well was quantified using Image J (http://imagej.nih.gov/ij /) and expressed as percentage response to different PVPA‐co‐AA treatments.
6
Quantitative Alkaline Phosphatase Assay in Osteoblasts
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For the measurement of alkaline phosphatase activity, one BCIP/NBT tablet (SigmaFastTM BCIP-NBT; Sigma Aldrich, St. Louis, MO, USA) was dissolved in 10 ml of distilled water and covered for the protection from light. A washing buffer was prepared by adding 0.05% Tween 20 to Dulbecco’s PBS without Ca++ and Mg++ (Thermo Fisher, Waltham, MA, USA)).
The MG-63 osteoblast-like cells were washed with PBS after the removal of the medium, and the membranes were covered with neutral buffered formalin (10%) for fixation. Afterwards, the membranes were washed using the washing buffer, and a volume of the BCIP/NBT substrate solution was added to fully cover the membranes. The plates were then incubated at room temperature in the dark for 10 min, the membranes were washed again using the washing buffer, and PBS was added to each well.
The presence of alkaline phosphatase activity on the membranes was detected by a change of colour to dark purple, which was analyzed through image analysis. For this, four micrographs were taken from each membrane. The membranes were divided in four quadrants and the area of the stained regions was measured using ImageJ 1.48 software (NIH, Bethesda, MD, USA).
The MG-63 osteoblast-like cells were washed with PBS after the removal of the medium, and the membranes were covered with neutral buffered formalin (10%) for fixation. Afterwards, the membranes were washed using the washing buffer, and a volume of the BCIP/NBT substrate solution was added to fully cover the membranes. The plates were then incubated at room temperature in the dark for 10 min, the membranes were washed again using the washing buffer, and PBS was added to each well.
The presence of alkaline phosphatase activity on the membranes was detected by a change of colour to dark purple, which was analyzed through image analysis. For this, four micrographs were taken from each membrane. The membranes were divided in four quadrants and the area of the stained regions was measured using ImageJ 1.48 software (NIH, Bethesda, MD, USA).
7
Influenza Antibody Quantification
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Antibody concentrations in BLF and antibody-producing B cells were monitored throughout the experiment by ELISA and ELISPOT assays, respectively. Total IgM was captured with purified anti-mouse IgM (Biolegend). Anti-influenza antibody was captured with inactivated influenza virus. For ELISA assay, all antibodies were detected with HRP-conjugated secondary anti-mouse IgM (Biolegend). TMB substrate (BioLegend) was used as a substrate for the HRP enzyme. For ELISPOT assay, all antibodies were detected with AP-conjugated anti-mouse IgM antibody (Biolegend). SIGMAFASTTM BCIP/NBT (Sigma–Aldrich) was used as a substrate for the AP enzyme.
8
Osteogenic and Chondrogenic Scaffold Evaluation
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All samples were fixed with 4% paraformaldehyde (PFA), rinsed with distilled water once, then stained with a 1% Alizarin Red S (AR) solution (pH 4.2 Sigma-Aldrich, Saint-Quentin Fallavier, France) for 20 min at room temperature. After the removal of AR solution, the samples were rinsed several times with distilled water. This staining was carried out on the scaffolds grown for 10 and 21 days in osteogenic or chondrogenic culture medium. An Alkaline Phosphatase Assay was performed, as well, on the hBM-MSCs grown for 7 days on the different scaffolds. Samples were first washed with PBS and then fixed with 10% formalin for 60 s. They were then washed with a washing buffer consisting of 0.05% Tween 20/Dulbecco’s PBS without Ca++/Mg++. Cells were stained using a BCIP (5-brom-4-chloro-3’-indolyphosphate p-toluidine salt)/NBT (nitro blue tetrazolium chloride) substrate solution (SigmaFastTM BCIP/NBT; Sigma Aldrich) at room temperature in the dark for 10 min. After the removal of the BCIP/NBT solution, the samples were washed with the washing buffer and then with PBS.
9
Osteogenic Differentiation of MSC
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The PromoCell protocol for Osteogenic Differentiation and Analysis of MSC was used for this assessment. To prepare the staining substrate solution, one BCIP/NBT tablet (SigmaFastTM BCIP-NBT; Sigma Aldrich) was dissolved in 10 ml distilled water. Washing buffer was prepared by adding 0.05% Tween 20 to PBS. The medium was aspirated from the wells, and the cells were washed. 10% neutral buffered formalin was added for 60 s, at which time the cells were again washed with buffer. The cells were then covered with BCIP/NBT substrate solution and incubated at room temperature in the dark for 10 min. The cells were then washed with washing buffer and s imaged for qualitative analysis or staining.
10
Osteoblast Differentiation Assay via ALP Staining
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Undifferentiated C3H10T1/2 cells show weak alkaline phosphatase (ALP) activity whereas differentiated osteoblasts show very high activity of ALP. ALP activity is therefore a good indicator of osteoblast differentiation. One BCIP/NBT tablet (SigmaFast TM BCIP-NBT, Sigma (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted December 18, 2020. ; https://doi.org/10.1101/2020.12.18.423369 doi: bioRxiv preprint Aldrich) was dissolved in 10ml distilled ddH20 to prepare the substrate solution which detects ALP by staining the cells blue-violet when ALP is present. It should be stored in the dark and used within 2hours. The washing buffer was made by adding 0.05% Tween 20 to PBS without Ca ++/ Mg ++ (Corning, #21-040-CM). Cells were washed one time with PBS and fixed with 4% paraformaldehyde for 60 seconds. After the fixation, cells were washed with washing buffer and then treated with BCIP/NBT substrate solution at room temperature in the dark for 10 minutes.
The staining process is being observed every 2-3 minutes. Cells are then washed with 1x with washing buffer and 1x with PBS respectively. After the last wash, the staining results were analyzed at the dissection microscope.
The copyright holder for this preprint this version posted December 18, 2020. ; https://doi.org/10.1101/2020.12.18.423369 doi: bioRxiv preprint Aldrich) was dissolved in 10ml distilled ddH20 to prepare the substrate solution which detects ALP by staining the cells blue-violet when ALP is present. It should be stored in the dark and used within 2hours. The washing buffer was made by adding 0.05% Tween 20 to PBS without Ca ++/ Mg ++ (Corning, #21-040-CM). Cells were washed one time with PBS and fixed with 4% paraformaldehyde for 60 seconds. After the fixation, cells were washed with washing buffer and then treated with BCIP/NBT substrate solution at room temperature in the dark for 10 minutes.
The staining process is being observed every 2-3 minutes. Cells are then washed with 1x with washing buffer and 1x with PBS respectively. After the last wash, the staining results were analyzed at the dissection microscope.
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