ROS production was determined by a quantitative nitro blue tetrazolium (NBT) assay as described previously (Serrander et al., 2007 (link)), with minor modifications. Briefly, cells grown in six-well plates were exposed to serum-free medium or LCM plus TGFβ treatment for 48 h and, after washing, incubated for 45 min in the corresponding medium supplemented with 1.67 mg/ml NBT. Cells were then fixed with ice-cold methanol and photographed with a Nikon Coolpix camera attached to Nikon eclipse TS100 microscope (40× objective), using the Nikon digital sight system and SPOT software. NBT was then extracted and solubilized using a mix of 560 μl of 2 M KOH and 480 μl of dimethyl sulfoxide. Absorbance of reduced NBT was measured at 630 nm. Data were normalized to protein levels determined in parallel samples.
Coolpix camera
Manufactured by Nikon
Sourced in Japan
The Nikon Coolpix camera is a compact digital camera designed for everyday use. It features an advanced image sensor and lens system to capture high-quality photographs. The camera's core function is to provide users with a convenient and portable device for capturing images.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e400 microscope, by Nikon (3 mentions)
Model cs 200 scale, by Ohaus (2 mentions)
Neomycin sulfate, by Merck Group (2 mentions)
Kanamycin sulfate, by Merck Group (2 mentions)
Tobramycin sulfate, by Merck Group (2 mentions)
Streptomycin sulfate, by Merck Group (2 mentions)
Gentamicin sulfate, by Merck Group (2 mentions)
Kanamycin disulfate, by Merck Group (2 mentions)
Synergy2 cam4, by Agilent Technologies (2 mentions)
Eclipse ts100, by Nikon (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Ts100 f phase contrast microscope, by Nikon (1 mentions)
Gelatin, by Thermo Fisher Scientific (1 mentions)
Zymogram sample buffer, by Bio-Rad (1 mentions)
Prism v6, by GraphPad (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
24 well microtiter plate, by Corning (1 mentions)
Ly 292 004, by Merck Group (1 mentions)
Sodium alginate, by Merck Group (1 mentions)
Scan software, by Epson (1 mentions)
24 protocols using coolpix camera
1
Quantitative Assay for ROS Production
2
Histological Analysis of Porcine Vocal Folds
3
Zymography Assay for Gelatinase Activity
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Sterile-filtered media were diluted 1:1 with zymogram sample buffer (BioRad) and 20 μl loaded into each well of a Tris-Glycine Novex 10% zymogram protein gel copolymerized with 0.1% gelatin (Invitrogen). SDS-Page gels were run at 118V constant voltage for 90 minutes. gelatinase activity was detected following the manufacturer’s protocol. Images of the gel demonstrating cleared regions of enzymatic activity were captured using a light box and Nikon CoolPix camera. Digital images were quantitated using ImageJ software [28 (link)]. NHDFs were cultured in CC-FGM with or without 50 μM of the MMP inhibitor GM6001 (http://www.emdmillipore.com ) for 72 hours.
4
Wound Measurement and Mouse Weighing
5
Microscopic Analysis of Living and Fixed Cells
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Living cells were imaged using a U-Plan-S-Apo 60× N.A. 1.42 objective lens mounted on an Olympus IX-81 spinning disc confocal microscope. The temperature was maintained using a custom-built heating system. Fixed cells were photographed on a Zeiss Axiophot microscope using a Zeiss 100x NA 1.4 PLAN-apochromat lens. Images were captured on a Nikon Coolpix camera. Level adjustment and cropping were performed using Adobe Photoshop CS6.
DAPI and Calcofluor staining was performed on cells that had been harvested by centrifugation, washed, and fixed with cold 70% (v/v) ethanol, as described previously [95 (link)]. Microscopy analysis of living cells was performed as described in [12 (link)], using the RodcellJ imageJ plugin [96 (link)]. Data were plotted using GraphPad Prism v6. In the whisker plots the box shows 25%-75% range for the population, the line indicates the median. The bars indicate 10% and 90% range for the population, and dots indicate more extreme individual values.
DAPI and Calcofluor staining was performed on cells that had been harvested by centrifugation, washed, and fixed with cold 70% (v/v) ethanol, as described previously [95 (link)]. Microscopy analysis of living cells was performed as described in [12 (link)], using the RodcellJ imageJ plugin [96 (link)]. Data were plotted using GraphPad Prism v6. In the whisker plots the box shows 25%-75% range for the population, the line indicates the median. The bars indicate 10% and 90% range for the population, and dots indicate more extreme individual values.
6
Colony and Pellicle Biofilm Formation
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For colony biofilm formation, cells were grown to exponential phase in LB broth and 2-μl of the culture was spotted onto LBGM [29 (link)] or MSgg [19 (link)] plates solidified with 1.5% agar (w/v). LBGM plates were incubated at 30°C for about 72 hrs and MSgg plates were incubated at 25°C for about 120 hrs. For pellicle biofilm formation, cells were grown to exponential phase in LB broth, and 2-μl of the culture was inoculated into 2-ml of LBGM or MSgg broth in a 24-well microtiter plate (Corning, NY, USA). LBGM pellicles were incubated for about 72 hrs at 30°C and MSgg pellicles were incubated for 48 hrs at 25°C. Images of colony and pellicle biofilms were taken using a Leica MSV269 dissecting scope or a Nikon Coolpix camera.
7
Bacterial Fixation and Microscopic Analysis
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Bacteria grown in sBHI to an OD620 of 0.4 were centrifuged and washed twice with phosphate-buffered saline (PBS). The final bacterial pellet was completely resuspended in 100 µl of PBS to remove all bacterial clumps and fixed by adding 100 µl of 4% paraformaldehyde. The sample suspension was placed on a microscope slide and visualized with a Leitz Dialux 22 light microscope with a Nikon Coolpix camera.
8
Wound Healing Assay for Migration Analysis
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Wound healing assay was used to detect cell migration ability. A2780, OVCAR3 and SK-OV-3 cells overexpressing miR-let-7b or control miRNA were seeded into 6-well culture plates and grown in DMEM or RMPI-1640 supplemented with 10% FBS until 90% confluence. Confluent cells were scratched with a pipette tip. Culture medium was removed and cells were rinsed with PBS, then incubated in DMEM or RMPI-1640 without FBS. Images were captured using a Nikon Coolpix camera at 0 and 24 h. The scratch area (SA) was measured using Image-Pro Plus 6.0 (NIH) and wound healing rate was calculated as follows: Wound healing rate=(initial SA-final SA)/initial SA.
9
Fibroblast-Macrophage Co-Culture Gel Contraction
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Gels were made in MatTek dishes for contraction assay as described20 (link). Orbital fibroblasts at a concentration of 1.5 × 105 cells/ml and macrophages of 2.25 × 105 cells/ml were used in co-culture experiments. The gels were photographed daily (Nikon Coolpix camera) for 7 days, and gel area was measured using ImageJ (https://imagej.nih.gov/ij/ ). The contraction was expressed as the percentage of gel area decrease compared to the original gel area.
10
Whole Mount Antibody Staining in Chick Embryos
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Whole mount antibody staining was performed as described previously [41 (link)]. In brief, the HH stage 8 embryos were removed from the yolk and washed rigorously with 1 × PBS to remove the yolk completely. The embryos were then fixed with 4% paraformaldehyde. 0.3% H2O2 solution made in 0.5% TBST was added to inactivate the endogenous peroxidase followed by 3 half an hour washes. Blocking solution was prepared with 3% BSA in TBST and embryos incubated with blocking solution for an hour. Primary antibodies (1:1000) were added in the blocking solution and the embryos incubated at room temperature for 2 days. Next secondary antibodies (1:2000) were added and the embryos incubated for 1 day. The colour development was carried out using a DAB substrate and H2O2. Photographs were taken at 4× magnification using Nikon cool pix camera adapted to a stereomicroscope.
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