A swarming motility assay was assessed as described previously [43 (link)]. Briefly, 3 μl of bacterial inoculum containing ~108 CFU/ml were placed in the center of 0.5% agar containing modified M9 medium with different concentrations of F-actin, DNA or Pf1 bacteriophage (0.005–0.1 mg/ml) in 6 well plates. Swarming area was evaluated from images captured with a Fuji Film LAS-300 system analyzed with Image Gauge (version 4.22) software.
Las 300 system
Manufactured by Fujifilm
Sourced in Japan
The LAS-300 system is a laboratory equipment designed for imaging and analysis. It provides high-quality image capture capabilities for a variety of samples and applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate system, by Thermo Fisher Scientific (1 mentions)
Proteome profiler mouse cytokine array panel a kit, by R&D Systems (1 mentions)
Gas permeable membrane, by USA Scientific (1 mentions)
7 protocols using las 300 system
1
Swarming Motility Assay with F-actin, DNA, and Phage
2
Cytokine and Angiogenesis Protein Profiling
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To identify proteins from the CM, 1 mL of the basal medium, low-glucose DMEM (10% FBS), and 1 mL of the TMSC-CM were each loaded onto cytokine and angiogenesis array membrane (R&D systems, Inc., Minneapolis, Minnesota, USA). After blocking the array membrane with blocking buffer for 1 h and membrane washing, the TMSC-CM and array detection antibody cocktail was mixed and added to the blocked membrane followed by overnight shaking incubation at 4°C. After washing, streptavidin-HRP buffer was added to the membrane, and incubation was performed for 30 min. Following another washing, Chemi Reagent Mixture was added to the membrane for reaction at room temperature for 1 min and measured using LAS-300 system (Fujifilm, Tokyo, Japan). Dot density was analyzed using Multi Gauge 3.0 software. Anti-inflammatory and pro-inflammatory cytokine levels were measured using cytokine array, and growth factor levels were measured using angiogenesis array.
3
Cell Lysate Protein Analysis
4
Western Blot Analysis of Brain Proteins
5
Cytokine Profiling in Colonic Tissue
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The levels of cytokines in the colonic tissue were analyzed using the Proteome Profiler Mouse Cytokine Array Panel A kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions. In brief, the tissues were lysed using radioimmunoprecipitation assay buffer (containing protease inhibitor cocktail). The protein concentration in the lysate was measured using a BCA Protein assay kit (Thermo Scientific, Waltham, MA, USA). Tissue protein diluted in array buffer was incubated with the ready-to-use pre-coated array membranes overnight at 4 °C on a rocking platform shaker. The membrane was washed and incubated with streptavidin–horseradish peroxidase (HRP) buffer for 30 min. Next, the membrane was washed and incubated with the Chemi Reagent mixture at 23–27 °C for 1 min. The membrane was analyzed using the LAS-300 system (Fujifilm, Tokyo, Japan). Dot density was analyzed using Multi Gauge 3.0.
6
Quantifying Bacterial Biofilm Formation
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The mass of biofilms was assessed using Crystal Violet (CV) staining (0.1%) [41 (link)] and chemiluminescence intensity measurements as an additional determinant of biofilm viability [42 ]. In each experiment an overnight culture of P. aeruginosa Xen 5, P. aeruginosa PAO1, P. aeruginosa P14 in TSB or S. aureus Xen 29, B. subtilis ATCC6051, E. coli MG1655, E. coli RS218 and C. albicans 1409 in LB was diluted to ~ 105 CFU/ml. When required, bacterial suspensions were placed on glass slides coated with poly-Lysine/F-actin or in flat bottom PVC microplates (MP Biomedicals; Solon, OH) sealed with a gas permeable membrane (USA Scientific; Ocala, FL). Bacteria or C. albicans were grown in 50% LB broth with or without F-actin, DNA, NFs, vimentin or Pf1 bacteriophage. A stationary biofilm was allowed to form for 24 or 48 h. PA Xen5 and S. aureus Xen29 chemiluminescence signals were evaluated using a Fuji Film LAS-300 system. Densitometry analysis was performed using Image Gauge (version 4.22) software.
7
Cell Lysate Protein Analysis
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After the desired cell treatment extraction of total cell lysates, SDS-polyacrylamide electrophoresis and immunoblot analysis were performed essentially as described previously (24 (link)). Enhanced chemoluminescence-based image acquisition was done on a Fuji LAS300 system coupled with densitometric analysis in the Multi Gauge software.
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