Lipofectamine 2000 and Lipofectamine RNAi MAX were purchased from Life Technologies (Carlsbad, CA). As primary antibodies, we used mouse monoclonal anti-CD59 (clone 5H8; Hirata et al., 2013 (link)), anti-FLAG (clone M2), anti-HA (clone HA7; Sigma-Aldrich, St. Louis, MO), anti-STX6 (Stressgen, San Diego, CA), anti–α-tubulin, anti-Golgin97 (clone CDF4; Life Technologies), anti-TMEM87A (clone #772807; R&D systems, Minneapolis, MN), rabbit monoclonal anti-LAMP1 (clone D2D11; Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-VPS53, VPS52 (a kind gift from Chris Schindler and Juan Bonifacino, National Institutes of Health, Bethesda, MD; Perez-Victoria et al., 2008 (link)), anti-GPP130 (Covance, Princeton, NJ), and anti-EEA1. The secondary antibodies were phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (IgG; BioLegend, San Diego, CA), Alexa Fluor 594–conjugated goat anti-mouse IgG, and Alexa Fluor 647–conjugated goat anti-mouse IgG (Life Technologies). Alexa Fluor 488–conjugated CTxB was purchased from Life Technologies.
Alexa fluor 647 conjugated goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Spain, United States
Alexa Fluor 647 conjugated goat anti-mouse IgG is a secondary antibody used for detection and visualization in various immunoassays and imaging techniques. It consists of a goat-derived polyclonal antibody specific to mouse immunoglobulin G (IgG), conjugated to the fluorescent dye Alexa Fluor 647.
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Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (6 mentions)
Macsquant analyzer, by Miltenyi Biotec (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 conjugated goat anti rat igg, by Thermo Fisher Scientific (3 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (3 mentions)
Facsaria, by BD (2 mentions)
Facscanto flow cytometer, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 conjugated donkey anti goat igg, by Thermo Fisher Scientific (2 mentions)
Pi plc, by Thermo Fisher Scientific (2 mentions)
D6w5b, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Cellquest pro 4, by BD (2 mentions)
Facsarray, by BD (2 mentions)
Saponin, by Merck Group (2 mentions)
Bz x700 microscope, by Keyence (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
40 protocols using alexa fluor 647 conjugated goat anti mouse igg
1
Antibody Labeling and Cell Organelle Markers
2
Nanog-GFP+ iPSC Isolation and Characterization
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Nanog-GFP-positive and DAPI-staining-negative single iPS cells were plated into each well of 96-well plates after being sorted by a FACS Aria instrument (BD). Seven days after plating, the cells were fixed and stained with an anti-Oct3/4 antibody (1∶100, C-10, Santa Cruz Biotechnology) and DAPI (Sigma-Aldrich). The secondary antibody used was Alexa Fluor 647-conjugated goat anti-mouse IgG (1∶500, Life Technologies). Subsequently, the number of Nanog-GFP positive colonies was counted.
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Immunolabeling Periostin and β1-integrin in Skin
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Frozen, 4% paraformaldehyde fixed and 0.25% TritonX-100 (Sigma Aldrich, Saint Louis, Missouri, USA) permeabilized 6 µm skin sections and were blocked with 3% normal goat serum and 1% bovine serum albumin containing (both Sigma Aldrich, Saint Louis, Missouri, USA) Tris-buffered saline. For immunolabeling mouse anti-human periostin (1:125, #sc‐398631, Santa Cruz Biotechnology), and β1-integrin (1:100, #ab30394, Abcam, Cambridge, UK) were used overnight followed by Alexa Fluor 647 conjugated goat anti-mouse IgG (Life Technologies, Carlsbad, California, USA). As isotype control mouse IgG1κ (#400102, BioLegend, San Diego, California, USA) was used, 4ʹ,6-diamidino-2-phenylindole (DAPI, 1:100, Sigma Aldrich) labeled the nuclei. Visualization, image processing and fluorescence quantification were performed by Zeiss Axio Imager Z1 microscope, ZEN 2012 Microscope Imaging software (Carl Zeiss AG, Oberkochen, Germany) and Fiji software (ImageJ, Wisconsin, USA).
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Quantifying Receptor Expression by Flow Cytometry
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For assessment of receptor expression levels by flow cytometry, cells (2 × 105) in conical-bottom 96-well plates were incubated for 1 h at 4°C in 0.1 ml final volume of FACS buffer (phosphate-buffered saline (PBS), 1% BSA, 0.1% NaN3) containing unconjugated anti-Flag mAb M2 (usually 2 μg/ml) or anti-CCR5 mAb 2D7 (2.5 μg/ml). In Fig 3F , the concentrations of unconjugated mAbs (M2, 2D7 and 45531) were saturating (40 μg/ml). After two washing steps in ice-cold FACS buffer, cells were incubated at 4°C for 30 min in 0.1 ml FACS buffer containing AlexaFluor 647-conjugated goat anti-mouse IgG (Life Technologies) at a 1:500 dilution. Cells were then washed twice in the FACS buffer and fixed with 2% paraformaldehyde-containing PBS. Data were acquired out on a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software. In the particular case of immunostaining of MDMs, 1.5 × 105 cells in 50 μl of FACS buffer were first incubated for 1 h at 4°C in the presence of 10% human serum AB (hSAB). Cells were then further incubated at 4°C for 20 min in 30 μl of FACS buffer in the presence of FcR blocker (2 μl / 106 cells, Myltenyi Biotec.). Then, labeling of receptors was carried out and analyzed as described above.
5
Tetramer-based Flow Cytometry Analysis
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Tetramer‐positive populations within PBMC were analysed using multi‐colour flow cytometry. In brief, PBMC (1 × 107/ml) were stained with phycoerythrin‐labelled tetramers at a final concentration of 20 nm for 30 min at room temperature, washed twice in PBS containing 0·5% FBS before staining with a mixture of monoclonal antibodies against CD4 (IL‐A12), TCR‐γδ (GB21A), CD21 (CC21, IgG116 ), NKp46 (EC1.1 IgG117 ) and CD172a (IL‐A24, IgG118 ) for 30 min at 4°. After three washes, cells were incubated for 30 min at 4° with Alexa Fluor 647conjugated goat anti‐mouse IgG (Life Technologies, Paisley, UK), washed three times, and then SYTOX Red Dead Cell Stain (Life Technologies) was added. Flow cytometric analysis was then conducted on a FACSCalibur or cell‐sorting on a FACSAria (both BD Biosciences, Oxford, UK). All monoclonal antibodies had been titrated to determine optimal dilutions before the experiment and the combination of antibodies and stains had been demonstrated to leave viable CD8 αβ T cells as the only unstained population (data not shown), which could therefore be gated as the Alexfluor647/SYTOX Red‐negative subset. For tetramer staining of cultured CD8 T cells staining with lineage‐specific antibodies was excluded.
6
Mapping Critical MAYV E2 Epitopes
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A pcDNA3.1(+) plasmid expressing a codon-optimized MAYV-TRVL4675 structural polyprotein (C, E3, E2, 6K, and E1 genes) was synthesized and mutated by Genewiz. Alanine scanning mutagenesis was performed on amino acids in the B domain of the E2 protein (residues 189–231), whereas charge mutations to arginine were made on residues 179–186 and 212–218, with the exception of 215, which was changed to glutamic acid. Plasmids were transfected into HEK-293T cells using Lipofectamine 3000 (Thermo Fisher Scientific). 18 h later, cells were chilled to 4°C, washed with PBS, and incubated with anti-MAYV mAbs (10 µg/ml) in PBS with 2% FBS for 1 h at 4°C. An oligoclonal mixture of all the anti-MAYV mAbs was used as a control for mutant E2 protein expression. Anti-MAYV mAb binding was detected using Alexa Fluor 647–conjugated goat anti-mouse IgG (Thermo Fisher Scientific) diluted 1:1,000. After 1 h, cells were washed, fixed with 1% PFA in PBS and analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec). Using previously described criteria (Smith et al., 2015 (link)), critical residues were defined as those with ≤25% binding to an individual mAb but ≥75% binding to an oligoclonal pool of anti-MAYV mAbs when detected by flow cytometry.
7
Mapping Critical Epitopes on Mayaro Virus Envelope Protein
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A pcDNA3.1(+) plasmid expressing a codon-optimized MAYV (strain TRVL4675) structural polyprotein (C, E3, E2, 6K, and E1 genes) was synthesized and mutated by GenScript. Alanine scanning mutagenesis was performed on amino acids in the A domain of the E2 protein (residues 1–173) that were predicted to be solvent exposed. Plasmids were transfected into HEK293T cells using Lipofectamine 3000 (Thermo Fisher). Eighteen hours later, cells were chilled to 4°C, washed with PBS, and incubated with anti-MAYV mAbs (10 μg/ml) in PBS with 2% FBS for 1 h at 4°C. An oligoclonal mixture of the 13 mAbs as well as an anti-B domain mAb (MAY-117) was used as a control for mutant E2 protein expression. Anti-MAYV mAb binding was detected using Alexa Fluor 647 conjugated goat anti-mouse IgG (Thermo Fisher) diluted 1:1000. After 1 h, cells were washed, fixed with 1% PFA in PBS, and analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec). Using previously described criteria (Smith et al., 2015 (link)), critical residues were defined as those with ≤25% binding to an individual mAb but ≥ 75% binding to an oligoclonal pool of anti-MAYV mAbs as determined by flow cytometry.
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Quantifying EGFR Expression in Tumor Xenografts
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Western Blot and Immunocytochemistry of ROC15 and HA-CETL
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Western blot analysis was performed as previously described [27 (link)]. Polyacrylamide gel (12.5%) (e-PAGEL, ATTO Corporation, Tokyo, Japan) was used for the observation of light- induced phosphorylation of ROC15 (Figs 5B , S9B and S9C ) and 7.5% polyacrylamide gel (e-PAGEL, ATTO Corporation) was used for the detection of HA-CETL (Figs 3B and S7E ). The transfer step was performed using a Qblot kit, EZ blot kit and EZFastBlot HMW kit (ATTO Corporation)). BLOCK ACE Powder (KAC, Kyoto, Japan) was used to block the membranes. Rat monoclonal anti-HA antibody was used as the primary antibody (1:10000, clone 3F10, Roche, Basel, Switzerland). Horseradish peroxidase-conjugated goat anti-rat IgG was used as a secondary antibody (1:25000, Merck KGaA, Darmstadt, Germany).
Immunocytochemistry was performed as described previously [27 (link)]. Rat monoclonal anti-HA (1:1000) and mouse monoclonal anti-NPC (1:1000, clone MAb414, Labcorp, NC, USA) were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rat IgG (Thermo Fisher Scientific) and Alexa Fluor 647-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) were used as secondary antibodies. Fluorescence was observed using a laser scanning confocal fluorescence microscope (FV10i-DOC; Olympus, Tokyo, Japan).
Immunocytochemistry was performed as described previously [27 (link)]. Rat monoclonal anti-HA (1:1000) and mouse monoclonal anti-NPC (1:1000, clone MAb414, Labcorp, NC, USA) were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rat IgG (Thermo Fisher Scientific) and Alexa Fluor 647-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) were used as secondary antibodies. Fluorescence was observed using a laser scanning confocal fluorescence microscope (FV10i-DOC; Olympus, Tokyo, Japan).
10
Flow Cytometry Analysis of Cell Surface Markers
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For flow cytometry, 2 × 105 SKOV-3 or Colo-320 cells were incubated with the respective primary antibodies, anti-CD81 (5A6), anti-CD9 (PAINS10), anti-β1 integrin (TS2/16), anti-α5 integrin (P1D6) and anti-ALCAM (PAINS-15) for 30 min at 4 °C, washed three times in FBS-free RPMI-1640 and incubated with the secondary polyclonal antibody Alexa Fluor™ 647-conjugated Goat anti-mouse IgG (Thermo Fisher, Madrid, Spain) for 30 min at 4 °C. Cells were washed three times and then fixed in a 2% formaldehyde solution in PBS. Fluorescence was measured using a FACScan™ flow cytometer (Beckton-Dickinson, Madrid, Spain). The cytometry data were processed using the FlowJo (v10 version) software (BD Biosciences, Ashland, OR, USA).
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