On day 9 of differentiation, media was changed to FBS-free media containing M-CSF and RANKL. Conditioned media was collected after 24 h, centrifuged, mixed with 2 volumes of Zymogram sample buffer (Bio-Rad) and loaded onto a 10% acrylamide gel containing co-polymerised gelatin (ZY00100BOX; ThermoFisher Scientific). Active recombinant human MMP8 and MMP9 (R&D systems) were used as positive controls. Gels were washed in renaturation buffer (BioRad) and then in development buffer (50 mM Tris, 10 mM CaCl2, 50 mM NaCl, 0.05% Brij 35, pH 7.6) to activate MMPs. Gels were processed for silver staining and scanned using an Epson V700 Photo scanner and Epsonscan software.
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Lab products found in correlation
Renaturation buffer, by Bio-Rad (2 mentions)
Zymogram sample buffer, by Bio-Rad (2 mentions)
Zy00100box, by Thermo Fisher Scientific (2 mentions)
Select agar, by Thermo Fisher Scientific (2 mentions)
Tris glycine gel electrophoresis, by Thermo Fisher Scientific (2 mentions)
Agarose, by Merck Group (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Rotor, by Singer Instruments (1 mentions)
15 protocols using v700 photo scanner
1
Zymography of Osteoclast-Derived MMPs
2
Zymography for Evaluating MMP Isoenzymes
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Zymography was used to assess the activity of different MMP isoenzymes. Proteins are separated in sodium dodecyl sulphate (SDS). After 9 days of osteoclast differentiation on acellular cartilage, the media was changed to FBS-free media with M-CSF and RANKL for 24 h. Conditioned media was collected, centrifuged to remove cell debris, and mixed with 2 volumes of Zymogram sample buffer (1610764; Bio-Rad). Samples were loaded onto a 10% acrylamide gel containing copolymerised gelatin (ZY00100BOX; ThermoFisher Scientific) run at 150 V for approximately 2 h. Active recombinant human MMP8 (908-MP-010, R&D systems) and MMP9 (911-MP-010, R&D systems) were used as positive controls. Gels were washed in renaturation buffer (1610765, BioRad) to remove SDS and then in development buffer (50 mM Tris, 10 mM CaCl2, 50 mM NaCl, 0.05 % Brij 35, pH 7.6) to remove renaturation buffer and activate MMPs. Gels were then processed for silver staining and scanned using an Epson V700 Photo scanner and Epsonscan software.
3
LamPART Lateral Flow Biosensing
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The 10μL LamPART reaction was prepared the same as above except that no UDP quenchers or fluorophores were used and biotin-labeled FIP and FITC-labeled BIP were used. The reaction was incubated at 37°C for 30min followed by 65°C for 30min. The amplicons were detected using PCRD Flex Lateral Flow Strips (Abingdon Health, Cat. No.: FG-FD51676) following the manufacturer’s protocol. Specifically, the 10μL post-amplification reaction was added to 140μL extraction buffers provided in the kit. The lateral flow strip was then dipped inside the buffer for 10min. The strips were scanned using an Epson V700 Photo Scanner.
4
Rugose Coral Taxonomy from Kowala Quarry
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The studied material comprises two colonies of rugose corals coming from the Famennian beds of the Kowala Quarry in the Holy Cross Mountains, Poland (Fig. 1 ). The specimens were found by the staff and students of geology from Silesia University. Thus, the material is housed at the Faculty of Earth Sciences, University of Silesia, Sosnowiec, Poland, under the abbreviation GIUS3619. Following the standard methods used for the taxonomic determinations of rugose corals, the material was studied in serial transversal and longitudinal sections (thin sections and acetate peels). The sections of the coralla were photographed and scanned using a Zeiss Stereomicroscope Discovery V20 with a Canon D70 camera, and an Epson V700 Photo scanner in the mode for transparent materials and resolution of 3200 dpi.![]()
5
Electron Microscopy of Seminiferous Tubules
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The seminiferous tubules were fixed in loco with 2,5% glutaraldehyde (electron microscope grade) in 0,1 M phosphate buffer (PB) pH 7.2 for 3 h at room temperature. The tubules were then mounted between two layers of 1,5% agarose (Sigma) of about 2 mm in height, which was cut into small cubes 2 × 2 × 3 mm in size and postfixed in 2% osmium tetroxide in 0,1 M PB overnight at 4 °C. The samples were dehydrated in a graded ethanol series, and embedded in epoxy resin. Semithin section (1 μm) were stained with toluidine blue in borax and examined by light microscopy. Ultrathin section (70 nm) were cut using a diamond knife on a Reichert Ultracut ultramicrotome, mounted on a Cu/Rh grids (200 mesh), contrasted with uranyl acetate and lead citrate, examined and photographed with a Zeiss 902 transmission electron microscope operating at 80 kV. The exposed films were developed according to common photographic techniques, captured with an Epson V700 Photo scanner with a final resolution of 600dpi and appropriately calibrated for contrast and brightness (see Fig. 1 ).
6
Measuring Bacterial Spreading Motility
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Semi-solid LB medium was prepared by supplementing with 0.15%-0.25% (wt. vol. À1 ) select agar (Invitrogen), boiling and subsequent cooling to 30 C-40 C before pouring into square Petri dishes of appropriate size. After solidification, 2 μl of exponentially grown cultures were spotted on the plates and incubated in a moist environment overnight at room temperature. Plates were scanned using an Epson V700 Photo Scanner before the spreading halos could merge. For direct comparison, different strains were always incubated on the same plate. Swimming/spreading ability was measured as relative diameters compared to that of wildtype cells or other appropriate reference strains.
7
Bacterial Spreading Ability Assay
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Spreading ability in semi-solid environments was analyzed by placing 4 μl of an exponentially grown planktonic culture on soft agar plates [LB supplemented with 0.3% (w/v) agar and, if applicable, 50 μg ml–1 of kanamycin and 40 ng ml–1 of anhydrotetracyclin) followed by an incubation period of 24 h at room temperature. Strains that were to be directly compared were always placed on the same plate. Plates were scanned for documentation using an Epson V700 Photo Scanner.
8
Milk Protein Profiling by SDS-PAGE
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Processed and digested milk samples (0.5 mL) were placed directly from the freezer into boiling water for 5 min to inactivate enzymes. Aliquots (75 μL) were desalted using a Zeba Spin Desalting Column (Thermo-Scientific, Rockford, IL) and a ZIP Spin centrifuge (LW Scientific, Lawrenceville, GA) operating at 1,500 × g for 2 min. Samples were prepared for SDS-PAGE and separated using a Criterion Cell and Basic PowerPac power supply according to manufacturer's instructions (Bio Rad, Hercules, CA); all gels, buffers, standards, and stains were obtained from BioRad. Briefly, samples were diluted 1:1 with tricine sample buffer containing 0.02% mercaptoethanol and boiled for 5 min. Samples (15 μL for gastric digests, 20 μL for intestinal digests, 10 μL for Precision Plus Dual Xtra Standards) were placed in the wells of a 10 to 20% gradient Criterion Tris-Tricine gel paired with Tris-Tricine-SDS running buffer, and separated at 150 V for 65 min. Gels were fixed for 30 min in acetic acid-methanol (10%:40%), stained for 30 min in Bio-Safe Coomassie stain, and destained in distilled water. Gels were scanned using a V700 Photo scanner (Epson America, Inc., Long Beach, CA) and protein profiles analyzed using densitometer software (Image Quant TL version 2003, GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
9
High-throughput Phenotyping of Yeast Strains
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Growth characteristics of strain collections were determined based on liquid microcultures performed in 384-well plates using the GATHODE software.6 (link) At least three biological replicates were used for each measured growth parameter. Trait profile analysis of the natural isolate collection was performed on solid media. Using a 384 matrix, cells were transferred from a YPD master plate to 26 different conditions (Table S4 ) using a pinning robot (RoToR, Singer instruments). After 48 h incubation at 30 °C, plates were scanned with the EPSON V700 Photo scanner at a resolution of 300 dpi. Size and circularity of the colonies were calculated for six biological replicates using the R package Gitter.53 (link) Using a similar procedure, trait variation within the “sake × tecc” segregating population was estimated on 12 conditions using 3 biological replicates (Tables S5 ).
10
Visualization of LDH Isozyme Separation
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LDH isozymes were separated by native gel electrophoresis at 125V for 1.25 hours using pre-cast 1 mm 6% Tris-glycine gel electrophoresis (Life Technologies, Carlsbad, CA, USA). Running buffer consisted of 25 mM Tris, 192 mM glycine, pH 8.3. After electrophoresis, the gel was soaked in 10 mL of 50mM Tris buffer, pH 8.0, containing LDH inhibitor at the specified concentrations for 30 min, followed by addition of reaction mixture [1 mg/mL iodonitrotetrazolium bromide (INT), 1 mM NAD+, 0.184 mg/mL phenazine methosulfate (PMS), and 5mM L-lactate], and gels were incubated on a rocker in the dark at 42°C for 7–14 minutes. After development of LDH activity bands, gels were destained with several rinses of de-ionized water in the dark until the background was clear. Finally, gels were scanned using an Epson V700 photo scanner in transmitted light mode to generate the images shown.
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