J774A.1 cells were stimulated as described above. After washing with PBS, the cells were fixed and permeated with 70–30 acetone–methanol as previously described (45 (link)). Briefly, cells were placed in a humidified slide chamber blocked for 1 h in 10% normal donkey serum (Jackson Immunologicals). After removal of the donkey serum, the primary antibodies to NLRP3 (Cryo-2; AdipoGen), caspase-1 (sc-514; Santa Cruz Biotechnology), and ASC (D2W8U; Cell Signaling) or isomolar, species-specific anti-IgG (R&D Systems, Inc.) were added and incubated overnight at 4 °C. The slides were then washed three times with PBS, and cells were incubated for 1 h at room temperature with donkey anti-mouse–Alexa555 (Life Technologies) or donkey anti-rabbit–Alexa488 (Life Technologies) conjugated secondary antibodies. Nuclei were stained with DAPI (Life Technologies). FRET images were acquired using a Marianas Imaging Station (Intelligent Imaging Innovations) using a Zeiss 639 Plan-Apochromat objective (1.4 N.A.), a Sutter Xenon light source, and a Cooke SensiCam (The Cooke Corporation) as previously described (45 (link)). The FRET was calculated as FRET = Transfer − (Fd/D) − (Fa/Aa) as reported (46 (link)).
D2w8u
Manufactured by Cell Signaling Technology
Sourced in United States
The D2W8U is a laboratory equipment product manufactured by Cell Signaling Technology. It serves as a core function to aid in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo pvdf transfer pack, by Bio-Rad (2 mentions)
Ab1872, by Abcam (2 mentions)
Ab214185, by Abcam (2 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Sc 514, by Santa Cruz Biotechnology (1 mentions)
Donkey anti mouse alexa 555, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cryo 2, by Adipogen (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab97040, by Abcam (1 mentions)
Ab227430, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
A5316, by Merck Group (1 mentions)
Lc3a b d3u4c, by Cell Signaling Technology (1 mentions)
D6b6s, by Cell Signaling Technology (1 mentions)
Antibodies against β actin ab8226, by Abcam (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
4 protocols using d2w8u
1
Immunofluorescence Imaging of NLRP3, Caspase-1, and ASC
2
Western Blot Analysis of Inflammasome Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed as previously described [23 , 24 ]. Proteins were quantified with the Bradford assay. Proteins were separated onto a pre-cast 4–20% polyacrylamide gel (Mini-PROTEAN® TGX gel, Biorad) and transferred to PVDF membranes (Trans-Blot® TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris–HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (ab8227, Abcam), nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing protein 3 (NLRP3) (ab214185, Abcam), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (D2W8U, Cell Signaling), caspase-1 (ab1872, Abcam), caspase-4 (#4450, Cell Signaling), caspase-5 (#46680, Cell Signaling), and caspase-8 (ab227430, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad). Densitometry was performed by the IBright Analysis software.
3
Colonic Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The colonic tissues were weighed and homogenised in lysis buffer, using a polytron homogeniser, as described by Richter et al. [60 (link)]. Proteins were quantified with the Bradford assay. Proteins (30 μg) were separated onto a pre-cast 4-20% polyacrylamide gel (Mini-PROTEAN® TGX gel, Biorad) and transferred to PVDF membranes (Trans-Blot® TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (monoclonal, diluted 1:5000, A5316, Sigma Aldrich), nucleotide-binding oligomerisation domain leucine rich repeat and pyrin domain containing protein 3 (NLRP3) (polyclonal, diluted 1:1000, ab214185, Abcam), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (monoclonal, diluted 1:1000, D2W8U, Cell Signaling) and caspase-1 (polyclonal, diluted 1:1000, ab1872, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040, Abcam, and anti-rabbit ab6721, Abcam). Protein bands were detected with ECL reagents (Clarity™ Western ECL Blotting Substrate, Biorad). Densitometry was performed by ImageJ software.
4
Libertellenone C Protects Against Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Libertellenone C (LC) was obtained as a white powder from the fermentation products of A. arundinis, which was analyzed by HR-MALDI mass spectrometry for the molecular formula C20H28O5 (obsd [M + Na]+ at m/z 371.1829, calcd [M + Na]+ 371.1834). LC was dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, MO, USA), with a stock concentration of 40 mM. H2O2 and sodium glutamate were purchased from RHAWN reagent (Shanghai, China). Antibodies against p-NF-κB p65 (SC-136548) and NF-κB p65 (SC-8008) were obtained from Santa Cruz Biotecnology, USA. Antibodies against NLRP3 (D2P5E, #13158), ASC (D2W8U, #67824), Caspase-1 (E2Z1C, #24232), iNOS (D6B6S, #13120), COX-2 (D5H5, #12282), HO-1 (E8B7A, #26416), and LC3A/B (D3U4C, #12741) were obtained from CST (Cell Signaling Technology, MA, USA). Antibodies against β-actin (Ab8226) were obtained from Abcam (Cambridge, UK).
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!