Tissues and cells were lysed as previously described (Bettaieb et al., 2014 ; Vazquez-Prieto et al., 2012 ). Aliquots of total homogenates containing 25–40 μg protein were denatured with Laemmli buffer, separated by reducing 7.5–12.5% polyacrylamide gel electrophoresis, and electroblotted onto PVDF membranes. Membranes were blocked for 1 h in 5% (w/v) bovine serum albumin and subsequently incubated in the presence of the corresponding primary antibodies (1:1000 dilution) overnight at 4 °C. After incubation for 1 h at room temperature in the presence of secondary antibodies (HRP conjugated) (1:10,000 dilution) the conjugates were visualized using enhanced chemiluminescence (ECL, Amersham Biosciences) and pixel intensities of immuno-reactive bands were quantified using FluorChem 8900 (Alpha Innotech). Data for phosphorylated proteins are presented as phosphorylation level normalized to protein expression.
Fluorchem 8900
Manufactured by Bio-Techne
Sourced in United States, United Kingdom
The FluorChem 8900 is a high-performance imaging system designed for a variety of fluorescence-based applications. It features a multi-channel detection system, advanced optics, and a sensitive CCD camera for capturing high-quality images. The FluorChem 8900 is capable of imaging a wide range of fluorescent samples, including gels, blots, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Cell Signaling Technology (3 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (2 mentions)
Anti phospho mek1 2, by Cell Signaling Technology (2 mentions)
Anti phospho p65, by Cell Signaling Technology (2 mentions)
C fos, by Cell Signaling Technology (2 mentions)
Anti phospho epha2, by Cell Signaling Technology (2 mentions)
Anti phospho c fos, by Cell Signaling Technology (2 mentions)
Ephb2, by Cell Signaling Technology (2 mentions)
Epha4, by Santa Cruz Biotechnology (2 mentions)
Epha2, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Mek1 2, by Cell Signaling Technology (2 mentions)
Anti phospho stat3, by Cell Signaling Technology (2 mentions)
Ezna bacterial dna isolation kit, by Omega Bio-Tek (2 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Irs 1, by Merck Group (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Proteome profiler array, by R&D Systems (1 mentions)
23 protocols using fluorchem 8900
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of Phosphorylated Proteins
3
Quantifying Cell Colony Formation
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Colony formation assay was performed as described previously [27 (link)]. Briefly, transfected cells were plated at a density of 1000 cells per well in 6-well plates and incubated for 14 days at 37 °C with 5% CO2. After washing with 4 °C pre-cooled PBS, cultures were fixed with ice-cold methanol for 20 min and stained with hematoxylin for 15 min. Colonies were photographed and calculated using a FluorChem 8900 image analysis system (Alpha Innotech, Miami, FL, USA). Each experiment was performed in triplicate.
4
Bacterial DNA Damage Quantification
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S. enterica wild-type, ΔdndB and ΔdndB–H, or E. coli B7A wild-type and ΔdndB–H were prepared as described for the survival assay. After 60 min of growth, cells were placed on ice and exposed to various doses of HOCl or H2O2. HOCl dependent strand-breaks were first observed at 7.5-fold×LD50 for each wild-type strain (see Supplementary Table 3 ), and these doses were then used for each corresponding ΔB–H strain. Effects caused by the genetic background of the strains and by the resulting difference in LD50 doses were ruled out by exposure of all bacterial strains to higher doses (25-fold×LD50 see Supplementary Table 3 ). Cells (in 1 mL LB) were pelleted at 8000×g for 2 min at 4 °C. Using reagents from the EZNA Bacterial DNA Isolation Kit (Omega Biotek), DNA was isolated by adding 2 μL lysozyme and 20 μL BTL digestion buffer for 10 min at 37 °C, followed by 20 μL proteinase K for 40 min at 55 °C and then 2 μL RNaseA for 2 min at 24 °C. The digestion mix was filtered through a 0.2 μm spin filter at 10,000×g for 10 min to remove insoluble cellular debris. A 40 μL aliquot was mixed with 5 μL of loading dye (6× dye from New England Biolabs) and loaded on a 0.7% agarose gel. A 1kb extended size marker from NEB was used. DNA was stained with ethidium bromide and visualized with FluorChem 8900 (Alpha Innotech).
5
Western Blot Analysis of Signaling Proteins
6
Profiling Immune Signaling in PBMC
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PBMC (4 × 10 [6 (link)]) were treated with C1q alone (25 μg/mL) [37 (link)], PfHz alone (10 μg/mL) and a combination of PfHz (10 μg/mL) + C1q (25 μg/mL) at 37 °C in complete RPMI 1640 medium. Cell lysates were harvested after C1q incubation (1 h) and PfHz incubation (4 h), followed by lysis in RIPA buffer containing protease and phosphatase inhibitors (Thermo Scientific) for 30 min on ice. Cell lysates were added to the human phosphor-immunoreceptor array membranes according to the manufacturer's protocol (Proteome Profiler Array; R&D Systems). Phosphorylated proteins were detected by pan-antiphosphotyrosine antibody conjugated to HRP. Array data were developed with X-ray film and quantified by FluorChem8900 (Alpha Innotech). Phosphorylation levels of individual analytes were determined by the average pixel density of duplicate spots; values were obtained after subtracting background signals and were normalised to positive controls. Culture supernatants were used for measurement of IL-1β, IL-6, TNF-α, LAIR1, and C1q protein levels through ELISA (see below).
7
Immunoblotting Analysis of Purified Ezrin Protein
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Purified Ezrin protein was processed by electrophoretic separation on 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membranes by the iBlot® 2 Dry Blotting System (Thermo Fisher Scientific). The PVDF membranes were incubated in blocking buffer (TBS containing 0.05% Tween-20 (TBST) and 5% nonfat dry milk) at room temperature for 1 h and were then incubated overnight at 4°C with a 1 : 100 dilution of serum and 1 : 2000 dilution of polyclonal anti-Ezrin antibody. We used HRP-conjugated goat anti-human/anti-mouse IgG as secondary antibodies at the dilution of 1 : 5000 which was recommended by the manufacturer. Finally, we detected immunoreactive bands by using the ECL kit (Thermo Fisher Scientific), which were photographed by FluorChem 8900 (Alpha Innotech, USA).
8
Cell Plate Colony Formation Assay
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Cell plate colony formation assays were performed as described previously [65 (link), 66 (link)]. Briefly, four cell lines (KYSE140, KYSE150, KYSE180 and TE3) were treated with 100 μM 5-aza-dC for 36 h before being trypsinized and plated into 6-well plates at a concentration of 2,000, 5,000 and 10,000 cells/well. The same four cell lines without 5-aza-dC treatment were used for the control. After washing with PBS, cultures were fixed with 4°C pre-cooled methanol for 15 min, and stained with hematoxylin for 15 min. Colonies were photographed and their sizes calculated with a FluorChem 8900 image analysis system (Alpha Innotech). Each experiment was performed in triplicate.
9
Bacterial Survival Assay
10
Protein Extraction and Western Blot Analysis
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Kidney cortex and harvested cultured cells were homogenized in lysis buffer supplemented with a protease inhibitor cocktail (Cell Signaling Technology, Inc., Beverly, MA, USA). Cytosolic protein fractions were obtained through incubation of cells with digitonin buffer [10 mM piperazine-N,N′-bis(2-ethanesulfonic acid), pH 6.8, 0.015% (wt/vol) digitonin, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride] for 10 min at 4°C (7 (link)). The supernatants of tissue, cell lysates and cytosolic protein extracts were extracted, subjected to protein assay and mixed with sodium dodecyl sulfate (SDS) loading buffer. Samples were loaded and separated by 12% SDS polyacrylamide gels (SDS-PAGE), electrotransferred onto a nitrocellulose membrane, blotted with the designated antibodies and then detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Amersham, UK). Densitometric quantification was performed with the image analysis program (Fluorchem™ 8900; Alpha Innotech, San Leandro, CA, USA).
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