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Oregon green 488 conjugated gelatin

Manufactured by Thermo Fisher Scientific
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Oregon Green 488-conjugated gelatin is a fluorescent-labeled protein that can be used for various applications in cell and molecular biology research. It serves as a tool for visualizing and tracking cellular structures or processes. The Oregon Green 488 dye provides a bright, green fluorescent signal that can be detected using standard fluorescence microscopy or flow cytometry techniques.

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29 protocols using oregon green 488 conjugated gelatin

1

Quantitative Cell Matrix Degradation Assay

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Coverslips (18 mm diameter) were prepared by subjecting to a 30% nitric acid wash (30 min) followed by 0.01% poly-L-lysine (Sigma-Aldrich) coating (15 min) and crosslinking with 0.5% glutaraldehyde (10 min, including extensive PBS washing). Gelatin coating was performed by incubating coverslips with pre-warmed 0.1% Oregon Green 488-conjugated gelatin (Invitrogen) and 0.2% porcine gelatin for 10 min to which 5 mg/ml NaBH4 (a reducing agent) was added (3 min). Coverslips were incubated in complete media for 2 h at 37°C before seeding (2 × 104 cells/ml) and overnight incubation. Cells were then fixed with 4% paraformaldehyde in 5% sucrose (20 min at 37°C), permeabilized in 0.5% Triton X-100-PBS (5 min), and immunolabelled (1:300; AlexaFluor 647-Phalloidin). Coverslips were mounted in Vectashield-mounting media (VectorLabs). Imaging was performed using Zeiss LSM 700 and Leica SP8 confocal microscopes. Quantitation of gelatin degradation was performed counting a minimum of 250 cells per variable. The total area of gelatin degradation was calculated using ImageJ. All assays were performed in triplicate.
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2

Visualizing Melanoma Cell Actin Cytoskeleton

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Coverslips were coated with warm Oregon Green® 488-conjugated gelatin (Invitrogen) as described in Ref(9 (link)). Melanoma cells (7.5 × 104) were seeded on the coverslips and after 16-hour incubation at 37°C they were fixed in 4% paraformaldehyde in PBS. After permeabilization in 0.05% triton x-100 in PBS, cells were stained with rhodamine-conjugated phalloidin (Invitrogen, to visualize actin) and hoechst (Thermo Scientific, to visualize nuclei). Coverslips were mounted onto glass slides with aqua-mount media (Polysciences, Warrington, PA). Fluorescent images were captured using a Nikon TE2000-E inverted microscope equipped with Roper CoolSnap HQ CCD camera and MetaVue software.
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3

Gelatin-coated cell culture and imaging

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12-well glass-bottomed culture plate (MatTek) was coated with Oregon Green 488-conjugated gelatin (Invitrogen; diluted 1:8 in solution of 2.5% (w/w) gelatin/2.5% (w/w) sucrose (Sigma-Aldrich)) for 10min at room temperature (RT). B16F10 cells were seeded (1.2x104 cells per well) and incubated at 37°C, 5% CO2. After 20 hours, cell cultures were fixed with 3.6% formaldehyde (Sigma-Aldrich; diluted in PBS), and permeabilized with 0.2% Triton-X100 (Sigma-Aldrich) in PBS for 15min at RT. Cell nuclei were visualized using Hoechst (1:1000 dilution, Fisher Scientific) by 1h incubation at RT. Stained samples were stored in PBS at 4°C until imaging.
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4

Quantifying Gelatin Degradation by Lymphocytes

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Glass coverslips (10 mm) were placed in a 24-well plate and precoated with 0.01% Poly-L-lysine (Sigma–Aldrich). The coverslips were then coated with 0.1% Oregon green 488-conjugated gelatin (Invitrogen), crosslinked with 0.5% glutaraldehyde, and coated with 2 μg/ml SDF-1α (PeproTech—RKP48061). Lymphocytes were incubated overnight over the gelatin-coated coverslips. Following incubation, cells were fixed and counterstained with phalloidin 594. Images were acquired with the Zeiss Confocal Laser Scanning Microscope (LSM 510 META) under a ×63 objective lens. The images were analyzed using ImageJ software, and the percentage of degradation was determined for each cell according to the equation: %Degradation area = (Area of degraded gelatin/Area of corresponding cells) × 100.
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5

Extracellular Matrix Degradation Assay

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ECM-degradation by cells on coverslips precoated with Oregon Green 488-conjugated-gelatin (Invitrogen) was analyzed for 18 h, as described in a previous report [22] (link).
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6

Visualizing Melanoma Cell Actin Cytoskeleton

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Coverslips were coated with warm Oregon Green® 488-conjugated gelatin (Invitrogen) as described in Ref(9 (link)). Melanoma cells (7.5 × 104) were seeded on the coverslips and after 16-hour incubation at 37°C they were fixed in 4% paraformaldehyde in PBS. After permeabilization in 0.05% triton x-100 in PBS, cells were stained with rhodamine-conjugated phalloidin (Invitrogen, to visualize actin) and hoechst (Thermo Scientific, to visualize nuclei). Coverslips were mounted onto glass slides with aqua-mount media (Polysciences, Warrington, PA). Fluorescent images were captured using a Nikon TE2000-E inverted microscope equipped with Roper CoolSnap HQ CCD camera and MetaVue software.
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7

Quantifying Osteoclast-Mediated Gelatin Degradation

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12-mm glass coverslips were coated with 10 µg/ml Oregon Green 488–conjugated gelatin (Invitrogen) in PBS for 30 min. Slides were washed with PBS and fixed with 4% PFA for 15 min. Residual PFA was removed by extensive washing in PBS and incubation with full medium for 30 min. 2 × 105 cells that were cultured in osteoclast differentiation medium for 24 h were seeded per coverslip. Cells were fixed with 4% PFA the following day and stained with DAPI. Five pictures were taken per slide with a Leica TCS SP5 X confocal microscope using a 40× NA 1.25–0.75 oil objective. Gelatin degradation was quantified by measuring the degraded area using ImageJ software and correlated to the number of nuclei.
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8

Fluorescent Gelatin-Coated Dishes for Invadopodia Assay

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Glass-bottomed dishes were treated with 0.1 mg/ml Poly L-lysine (Sigma-Aldrich) and crosslinked with 0.5% glutaraldehyde (Mecoxlane). Then, the dishes were coated with fluorescent gelatin [1:5 dilution of Oregon Green 488 conjugated gelatin (G13186, Invitrogen) with 0.1% unconjugated gelatin (G1393, Sigma)] at 37 ℃ for 1 h. The dishes were then incubated with 5mg/ml sodium borohydride for 15 min, and then incubated with 70% ethanol for 20 min. Three washes with 1×PBS were performed between each step. DMEM media was added to the dishes at 37 ℃ for 1 h before cell plating. After incubating the cells at 37 ℃ for a further 48 h, the cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100, rinsed with PBS and blocked in 3% bovine serum albumin (BAS) in PBS. The cells were then incubated with primary antibodies against Tks5/FISH (sc-30122, Santa Cruz) and F-actin/Phalloidin (A12380, Invitrogen), and then incubated with Alexa Fluor 647-labelled secondary antibodies (A-21245, Invitrogen). The samples were observed under a Nikon A1 confocal microscope. Quantification of the degraded gelatin area was performed using Image J software (National Institutes of Health).
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9

Quantifying Invadosome Formation

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Coverslips were prepared with Oregon green488 conjugated gelatin (Invitrogen, Burlington, ON, Canada) at a final concentration of 0.5%, as previously described [7 (link)]. Thirty thousand cells were seeded onto each coverslip, incubated for 48 h, and fixed with 4% paraformaldehyde. Nuclei were stained with DAPI, and F-actin was stained with Texas Red phalloidin. Stained cells were visualized using a Zeiss Axioskop fluorescence microscope, and invadosomes were identified by F-actin-enriched areas of matrix degradation. Three fields of 100 cells (magnification 40×) were counted per coverslip to quantify the percentage of invadosome-forming cells.
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10

Quantifying ECM-Degrading Invadopodia

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Coverslips were prepared as previously described [50 (link)], using Oregon-Green488-conjugated gelatin (Invitrogen, Burlington, ON, Canada). Forty thousand cells were seeded on each coverslip and allowed to adhere. Following various incubation times as described within the figure legends, cells were fixed with 2% paraformaldehyde for 10 min at room temperature. Nuclei were stained with DAPI and F-actin was stained using Texas-Red-conjugated phalloidin. Cells were visualized and imaged by fluorescence microscopy using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss, Inc., Thornwood, NY, USA). Cells forming ECM-degrading invadopodia were identified based on cells with at least one F-actin-enriched area of matrix degradation (characterized by loss of green fluorescence). Three fields of 100 cells (magnification 40×) were counted per coverslip to quantify the percentage of cells forming ECM-degrading invadopodia.
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