Total H. chilense genomic DNA was also labelled by nick-translation with biotin-11-dUTP (Boehringer Mannheim Biochemicals, Germany) or digoxigenin-11-dUTP (Roche Applied Science, Indianapolis, IN, USA) and used as a probe. The in situ hybridisation protocol was performed according to Prieto et al. (2004b (link)). The amount of either the biotin- or digoxigenin-labelled probes in the hybridisation mixture was 5 ng. Unlabelled wheat genomic DNA was used as blocking DNA at a ratio of 1:50 (probe/blocking DNA). Biotin-labelled H. chilense DNA and digoxigenin-labelled H. chilense DNA were detected with a streptavidin-Cy3 conjugate (Sigma, St. Louis, MO, USA) and antidigoxigenin-FITC (Roche Diagnostics, Meylan, France), respectively. Chromosomes were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). Hybridisation signals were visualised using a Nikon Eclipse 80i epifluorescence microscope. Images were captured with a Nikon CCD camera using the Nikon 3.0 software (Nikon Instruments Europe BV, Amstelveen, The Netherlands) and processed with Photoshop 4.0 software (Adobe Systems Inc., San Jose, CA, USA).
Streptavidin cy3 conjugate
Manufactured by Merck Group
Sourced in United States, Japan
Streptavidin-Cy3 conjugate is a molecular tool that combines the high-affinity binding of streptavidin to biotin with the fluorescent properties of the Cy3 dye. It is commonly used in various biotechnological and research applications that require the detection and visualization of biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Neurobiotin, by Vector Laboratories (2 mentions)
Pli 188, by Nikon (2 mentions)
Anti digoxigenin fitc, by Roche (1 mentions)
Digoxigenin 11 dutp, by Roche (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Matlab, by MathWorks (1 mentions)
Influx cell sorter, by BD (1 mentions)
Ficoll paque gradient, by GE Healthcare (1 mentions)
Iron 3 chloride fecl3, by Merck Group (1 mentions)
Millipore water, by Merck Group (1 mentions)
1 propanol, by Merck Group (1 mentions)
Ammonia solution, by Merck Group (1 mentions)
Iron 2 chloride, by Merck Group (1 mentions)
Neurobiotin, by Merck Group (1 mentions)
Lucifer yellow, by Merck Group (1 mentions)
Sigmaplot 10, by Grafiti LLC (1 mentions)
Lucifer yellow, by Thermo Fisher Scientific (1 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Neurobiotin dye, by Vector Laboratories (1 mentions)
Lucifer yellow dye, by Thermo Fisher Scientific (1 mentions)
7 protocols using streptavidin cy3 conjugate
1
In situ hybridization of H. chilense genomic DNA
2
Tracking GFP-L. major Parasites in Murine Lymphoid Tissue
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OCT-embedded lymph node and spleen tissue from mice infected with GFP-L. major IL81 parasites for 4 weeks was cut into 10 μm cryosections. Following acetone fixation, dendritic cells were stained using biotinylated anti-CD11c mAb (BD Biosciences) and visualized by staining with a streptavidin-Cy3 conjugate (Sigma). Nuclei were stained with Hoechst. Coverslips were then mounted on sections using Mowiol®4-88 mounting medium (Calbiochem) with anti-fade (Sigma). Images were acquired by Ziess LSM 510 confocal microscope and images quantified using a MatLab (MathWorks, Natick, Massachusetts) script developed for automated counting. A total of 16 fields were captured for each condition. At week 4 after GFP-L. major IL81 infection, lymph node B cells (CD19+CD3−CD11c−) were isolated by cell sorting on a FACS Vantage cell sorter. Sorted cells were viewed live, directly in suspension in chamber slides for the presence of GFP+ parasites by LSM 510 confocal microscopy.
3
Isolation of HA-Specific Memory B Cells
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This study was approved by the Ethics Review Committee of Institut Pasteur of Shanghai, CAS (IPS-2016004). The informed consent was obtained by the participant. Blood was collected from a female volunteer previously ~ 4 weeks after inoculating with seasonal split influenza vaccine after she had signed the informed consent form. The vaccine was produced by Shanghai Institute of Biological Products Co., Ltd. in 2016, which contains three components, A/California/7/2009(H1N1) pdm09-like virus, A/Victoria/361/2011(H3N2)-like virus and B/Wisconsin/1/2010-like virus. Fresh PBMCs were isolated from the collected blood by using the Ficoll-Paque gradient (GE Healthcare, cat. no. 17144002). The PBMCs were stained with a FITC-labeled mouse anti-human CD19 antibody (BD PharmingenTM, cat. no. 555412) and an APC-labeled mouse anti-human IgG antibody (BD PharmingenTM, cat. no. 550931). The recombinant HK/68 H3N2 HA protein was labeled with biotin using the EZ-Link Sμlfo-NHS-LC Biotin biotinylation reagent (Thermo Fisher Scientific, cat. no. 21335), which could bind to the streptavidin-Cy3 conjugate (Sigma-Aldrich, cat. no. S6402). Single FITC-CD19+/APC-IgG+/streptavidin-Cy3 conjugate-HA-specific memory B cells were isolated with a BD InfluxTM Cell Sorter and sorted into 96-well plates. The data were analyzed by FlowJo V10 software.
4
Synthesis of Magnetic Silica Nanoparticles
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All buffers and solutions were prepared using Millipore water (resistivity equal to 18.2 MΩ cm). Iron(ii ) chloride (FeCl2·4H2O), iron(iii ) chloride (FeCl3), 3-aminophenyl boronic acid monohydrate (APBA), bovine serum albumin (BSA), dextran (D-1662), and streptavidin-Cy3 conjugate were purchased from Sigma-Aldrich and used as received. Tetraethyl orthosilicate (TEOS) was purchased from Fluka. 25% Ammonia solution and 1-propanol were obtained from Merck. Anti-SAA mAb was purchased from Anogen. 3-(Aminopropyl)trimethoxysilane (APTS) was obtained from Sigma.
5
Quantifying Gap Junction Permeability
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Parental (untransfected) HeLa cells and HeLa cells stably transfected with wild-type Cx50, Cx50R33E, Cx50E162R or Cx50R33E,E162R were grown on glass coverslips until they reached 90‒95% confluence. Then, one cell within a cluster was microinjected with a solution containing 9% Neurobiotin (charge: + 1; MW: 287.2; Vector Laboratories, Burlingame, CA, USA) and 5% Lucifer yellow (charge − 2; MW: 444.4; Sigma-Aldrich, St. Louis, MO, USA) for 1 min using a picospritzer (model PLI-188; Nikon Instruments Inc., Melville, NY, USA) [63 (link)]. After allowing the microinjected gap junction tracers to diffuse to neighboring cells for 10 min, cells were fixed in 4% paraformaldehyde for 15 min and incubated with Cy3-streptavidin conjugate (Sigma-Aldrich) to detect Neurobiotin by fluorescence microscopy [63 (link)]. Lucifer yellow facilitated identification of the injected cell, because human Cx50 has limited permeability to this dye and did not spread [64 (link)]. The extent of Neurobiotin intercellular transfer was determined by counting the number of adjacent cells containing the tracer. The number of microinjections ranged from 10 to 24 for cells expressing the different constructs. Data are presented as mean ± S.E.M. Statistical analysis was performed using Student’s t-test. Graphs were generated in SigmaPlot 10 (Systat Software, Inc., Palo Alto, CA, USA).
6
Microinjection for Intercellular Tracer Transfer
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For microinjection studies, cells were cultured on glass coverslips and treated with EVs as described above. The coverslip was transferred to F-12 medium (Thermo Fisher Scientific Inc.) buffered with 15 mM HEPES. Individual cells within clusters were impaled and injected with 5% Lucifer yellow (charge = −2, MW = 456; Thermo Fisher Scientific Inc.) and 5% Neurobiotin (charge = +1, MW = 322.8; Vector Laboratories, Burlingame, CA dissolved in water) for 1 min using a picospritzer (model PLI-188, Nikon Instruments, Melville, NY, USA). Inclusion of the Lucifer yellow tracer allowed visualization of successful microinjection of the impaled cell under UV illumination. After injection, cells were fixed in 4% paraformaldehyde for 30 min and permeabilized for 2 min with methanol/acetone (1:1) at RT. The Neurobiotin tracer was detected via staining with Cy3-streptavidin conjugate (Sigma-Aldrich) at 1:1000 dilution for 30 min at RT. Intercellular transfer of tracers was quantified by counting the number of adjacent cells containing the tracer.
7
Intercellular Tracer Transfer Assay
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The cells were cultured with normal culture medium on glass coverslips until 80%–90% confluence was reached. Then, the glass coverslips were transferred to a bath solution containing 140 mM NaCl, 1 mM MgCl2, 5.4 mM KCl, 1.2 mM CaCl2, and 10 mM HEPES (pH 7.2). The cells were impaled with a micropipette filled with a patch pipette solution and 4% neurobiotin dye (Mr = 322.8, charge = +1; Vector Laboratories, Burlingame, CA, USA) or 5% lucifer yellow dye (Mr = 457, charge = −2; Invitrogen). The patch pipette solution contained 140 mM KCl, 1 mM MgCl2, 5 mM NaCl, and 10 mMHEPES (pH 7.4). The dye solution was microinjected for 3 min with a Picospritzer (model PLI-188; Nikon, Tokyo, Japan).
After neurobiotin dye injection, the cells were fixed, permeabilized, and stained with Cy3-streptavidin conjugate (Sigma) for tracer detection. However, after lucifer yellow dye injection, the cells required only fixation. Both types of cells were then photographed with fluorescence microscopy. The adjacent cells stained by the dye were counted to determine the extent of the intercellular tracer transfer. Three independent stable-cell clones were all used to perform the dye transfer assay. The number of coupled cells was expressed as the means ±SDs. Statistical analysis was performed with Student's t-test.
After neurobiotin dye injection, the cells were fixed, permeabilized, and stained with Cy3-streptavidin conjugate (Sigma) for tracer detection. However, after lucifer yellow dye injection, the cells required only fixation. Both types of cells were then photographed with fluorescence microscopy. The adjacent cells stained by the dye were counted to determine the extent of the intercellular tracer transfer. Three independent stable-cell clones were all used to perform the dye transfer assay. The number of coupled cells was expressed as the means ±SDs. Statistical analysis was performed with Student's t-test.
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