Media was aspirated from wells, cells were washed with DPBS, and lysed in the plate with RIPA (Sigma #R0278) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche #11836153001). Lysate was collected and centrifuged at 13,000g for 10 minutes at 4°C. BCA protein assay determined protein content and samples were normalized with RIPA/protease inhibitor. Samples were treated with Lambda Phosphatase (NEB #P0753L) for 3 hours at 30°C. LDS NuPAGE sample buffer with 5% β-mercaptoethanol was added and samples were heated to 95°C for 3 minutes then loaded onto a NuPAGE 8% 20-well Midi gel (Invitrogen WG1002), with Tau Peptide Ladder (rPeptideT-1007–2). After running, the gel was transferred onto either 0.45um PVDF membrane for 2hr at 400mA. Membrane was both blocked and treated with primary antibodies in TBS/0.1% Tween20 overnight at 4°C: rabbit anti-Tau antibody (Aligent/DAKO, A002401-2 1:1000-10,000). The next day, membrane was washed 3 times with TBST, then treated with peroxidase-conjugated goat anti-rabbit secondary antibodies in 5% Milk/TBS/0.1% Tween 20 for up to 3hrs at RT. Membrane was washed 3 times with TBS/0.1% Tween20 and developed with the ECL system (Thermo Scientific, #34076) and imaged on a Sapphire Imager (Azure Biosystems).
Sapphire imager
Manufactured by Azure Biosystems
Sourced in Germany
The Sapphire Imager is a high-performance imaging system designed for scientific research applications. It features a sensitive camera, versatile lighting options, and advanced image acquisition and analysis capabilities. The Sapphire Imager is capable of capturing detailed images of various samples, but its specific intended use is not provided in this description.
Automatically generated - may contain errors
Lab products found in correlation
Celltracker green cmfda dye, by Thermo Fisher Scientific (2 mentions)
Lambda phosphatase, by New England Biolabs (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Collagen g, by Merck Group (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Bis tris sds page gel, by Thermo Fisher Scientific (1 mentions)
Mes sds buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Fluoroblok insert, by Corning (1 mentions)
5 protocols using sapphire imager
1
Tau Protein Extraction and Western Blot
2
Adhesion of SHI-treated RCC Cells to ECM and HUVECs
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To investigate the interaction of control and 72 h SHI-treated cells with extracellular matrix proteins (ECM), 24-well tissue culture plates were pre-coated with collagen G (200 µg/mL; Merck, Darmstadt, Germany), fibronectin (10 µg/mL; Gibco™, Thermo Fisher Scientific, Darmstadt, Germany) or laminin (10 µg/mL; Corning GmbH, Kaiserslautern, Germany) overnight at 4 °C. Wells covered with PBS served as a background control for the unspecific binding. To minimize non-specific cell adhesion, plates were blocked 1 h before assay with 1% BSA.
To determine the interaction of tumor cells with the vascular endothelium, 1.25 × 105 HUVECs were plated into 24-well-plates 16 h prior to an adhesion assay. For adhesion assays 1.25 × 105 parental and sunitinib-resistant RCC cells, pre-stained with CellTracker Green CMFDA Dye (Invitrogen™, Thermo Fisher Scientific, Darmstadt, Germany) and pre-treated with SHI or diluent, were used per well and incubated for 1 h (adhesion to ECM) or 2 h (adhesion to HUVEC) at 37 °C in a humidified CO2 incubator. Non-attached tumor cells were washed off with PBS, containing Mg2+ and Ca2+, and fixed with 2% glutaraldehyde. To score adhesion, the mean fluorescent intensity of attached cells was determined using a Sapphire Imager (Azure Biosystems, Munich, Germany). Values were presented as a percentage compared to untreated controls, set to 100%.
To determine the interaction of tumor cells with the vascular endothelium, 1.25 × 105 HUVECs were plated into 24-well-plates 16 h prior to an adhesion assay. For adhesion assays 1.25 × 105 parental and sunitinib-resistant RCC cells, pre-stained with CellTracker Green CMFDA Dye (Invitrogen™, Thermo Fisher Scientific, Darmstadt, Germany) and pre-treated with SHI or diluent, were used per well and incubated for 1 h (adhesion to ECM) or 2 h (adhesion to HUVEC) at 37 °C in a humidified CO2 incubator. Non-attached tumor cells were washed off with PBS, containing Mg2+ and Ca2+, and fixed with 2% glutaraldehyde. To score adhesion, the mean fluorescent intensity of attached cells was determined using a Sapphire Imager (Azure Biosystems, Munich, Germany). Values were presented as a percentage compared to untreated controls, set to 100%.
3
Quantitative Western Blot Analysis
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Samples (25 mg of protein per well/lane) were separated by SDS-PAGE Bis-Tris gel (Invitrogen) and MES-SDS buffer (Invitrogen). All groups (Y, AU, and AI) were represented within each gel. Following separation, proteins were transferred onto PVDF membranes (Invitrogen) using an iBlot dry blotting system (Invitrogen). The antibodies were diluted in blocking solution (2% ELC advance blocking agent; GE Healthcare) in wash buffer (TBS and 0.1% Tween 20) and membranes were incubated overnight at 4°C. Immunoreactivity was detected by application of conjugated secondary antibodies (Alexa Fluor 488; Alexa Fluor 546; CY5; Jackson ImmunoResearch). The immunoblots were scanned on a Sapphire imager (Azure Biosystems) at 100-mm resolution and quantified using ImageJ software (National Institutes of Health). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin were used as loading controls and all results were normalized to the expression of these proteins.
4
In Vitro Kinase Assay for cGAS
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WT and mutant HA-SRC was incubated with FLAG-cGAS in kinase buffer (5 mM MOPS [pH 7.2], 2.5 mM β-glycerol-phosphate, 4 mM MgCl2, 2.5 mM MnCl2, 1 mM EGTA, 0.4 mM EDTA, 10 mM DTT, 50 μM ATP, 4 μCi γ-32P ATP) at 30°C for 30 minutes. Nonradioactive assays were conducted with 250 μM ATP. Endpoint assay reactions were stopped by adding 4X SDS-PAGE loading dye at 95°C for 5 minutes, followed by SDS-PAGE analysis. Radioactive SDS-PAGE gels were imaged on Azure Sapphire imager.
5
Chemotactic Activity and Cell Migration Assay
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Chemotactic activity and cell migration were assessed using Falcon® 24-well Companion plates, and Corning® FluoroBlok Inserts with 8 µm pore size (both: Corning GmbH, Kaiserslautern, Germany). To determine cell motility toward a soluble chemotactic agent, an FCS gradient was employed. For chemotaxis, inserts were left unaltered. To score cell migration, inserts were pre-coated with 200 µg/mL of collagen G overnight at 4 °C.
Aliquots of 6 × 105 parental and sunitinib-resistant RCC cells, pre-stained with CellTracker Green CMFDA Dye (Invitrogen™, Thermo Fisher Scientific, Darmstadt, Germany) and 72 h pre-treated with SHI or diluent, were seeded per insert. The companion plates were loaded with cell growth media containing 30% FCS and inserts with the cell suspension in media with 10% FCS. After 24 h incubation, the inserts were washed with PBS, containing Mg2+ and Ca2+, and fixed with 2% glutaraldehyde. To quantify the number of cells migrating through the insert membrane toward the 30% FCS stimulus, mean fluorescent intensity on the lower surface of the inserts was determined using a Sapphire Imager (Azure Biosystems, Munich, Germany). Values were presented as a percentage compared to untreated controls, set to 100%.
Aliquots of 6 × 105 parental and sunitinib-resistant RCC cells, pre-stained with CellTracker Green CMFDA Dye (Invitrogen™, Thermo Fisher Scientific, Darmstadt, Germany) and 72 h pre-treated with SHI or diluent, were seeded per insert. The companion plates were loaded with cell growth media containing 30% FCS and inserts with the cell suspension in media with 10% FCS. After 24 h incubation, the inserts were washed with PBS, containing Mg2+ and Ca2+, and fixed with 2% glutaraldehyde. To quantify the number of cells migrating through the insert membrane toward the 30% FCS stimulus, mean fluorescent intensity on the lower surface of the inserts was determined using a Sapphire Imager (Azure Biosystems, Munich, Germany). Values were presented as a percentage compared to untreated controls, set to 100%.
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