Human intestinal cell lines HT-29 and THP-1 human monocyte cells were purchased from ATCC. Wild type and TLR9 knock-out mouse macrophage cell lines were collected as we previously described 26 (link) and HEK293 wild type cell line were acquired from BEI Resources. TcdA was purified from C. difficile strain VPI 10463 (ATCC, Maryland, USA). Culture supernatant was fractionated by anion-exchange chromatography, and TcdA was isolated by precipitation in acetate buffer as described previously 27 (link). TcdA was labeled using an Alexa Fluor 555 Protein Labeling kit (Molecular Probes, Inc.) according to the manufacturer’s protocol. Briefly, TcdA in TE buffer was eluted through a PD-10 column prior equilibrated with PBS to remove Tris-HCL buffer, and 0.5ml of TcdA (1.7mg) in PBS was mixed with 50μl 1M sodium bicarbonate and 1 vial of reactive dye. The mixture was stirred at room temperature for 1 hour and unlabeled free dye was removed by resin column chromatography.
Alexa fluor 555 protein labeling kit
Manufactured by Thermo Fisher Scientific
The Alexa Fluor 555 protein labeling kit is a reagent used to fluorescently label proteins. It contains the Alexa Fluor 555 dye, which can be covalently attached to proteins, allowing for their visualization and detection in various applications.
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Lab products found in correlation
Alexa 647 streptavidin, by Jackson ImmunoResearch (2 mentions)
Hm1013, by Hycult Biotech (2 mentions)
Ab19808, by Abcam (2 mentions)
C6219, by Merck Group (2 mentions)
Ab11941, by Abcam (2 mentions)
Ab14055, by Abcam (2 mentions)
Affinipure minimal cross reactivity secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Af743, by R&D Systems (2 mentions)
Ht 29, by American Type Culture Collection (1 mentions)
Thp 1 human monocyte cells, by American Type Culture Collection (1 mentions)
Vpi10463, by American Type Culture Collection (1 mentions)
Exoview r100, by Unchained Labs (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Alexa 647, by Jackson ImmunoResearch (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Lyve 1, by Thermo Fisher Scientific (1 mentions)
Af3827, by R&D Systems (1 mentions)
A0082, by Agilent Technologies (1 mentions)
L9393, by Merck Group (1 mentions)
Ab5060, by Abcam (1 mentions)
10 protocols using alexa fluor 555 protein labeling kit
1
Fluorescent labeling of C. difficile toxin A
2
Extracellular Vesicle Characterization Using ExoView
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Particle distribution and concentration were obtained using ExoView R100 (Nanoview Biosciences) analysis as previously described [42 (link)]. Briefly, binding of EVs to the microarray chip (EV-TETRA-P) coated with different antibodies, including anti-CD9, anti-CD81, and anti-CD63, ensures measured particles express specific surface components. ExoView has a high sensitivity and can accurately measure single EV particles ∼50 nm diameter using single-particle interferometric reflectance imaging. About 2.5 μg EVs were diluted in 500 μl incubation solution and 40 μl placed on a chip to incubate overnight. Following three washes with incubation solution, the chips were then incubated with antibodies (CD9, CD81, and PLAP, 1:1200 dilution) at room temperature for 1 h followed by one wash in incubation solution and three washes with wash solution. Chips were rinsed and then analyzed (ExoView R100). Chips coated with each antibody were prepared in triplicate and the whole chip was scanned for particle analysis. Detection of Placental Alkaline Phosphatase (PLAP) on circulating plasma-derived sEVs was performed using anti-PLAP, labeled using Alexa Fluor 555 Protein Labeling Kit (A20174, Molecular Probes) according to the manufacturer’s instructions.
3
UDF Labeling and Binding Assay
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The concentrated Superdex 200 fraction of UDF (2 mg/mL) was labeled with an Alexa Fluor 555 protein labeling Kit (Molecular Probes) according to the manufacturer’s instructions. Spermatids and spermatozoa were incubated with Alexa 555-UDF at 37 °C for 20 min and then washed with HKB buffer. Samples were examined with an LSM710 confocal microscope (Carl Zeiss Microscopy, Oberkochen, Germany). The fluorescence intensities of samples were also measured with flowcytometry. In competition assay spermatozoa were incubated with unlabeled UDF at 37 °C for 20 min and then centrifuged at 2000 rpm for 1 min. The supernatant was removed and cells were rinsed in HKB buffer (pH 7.1) and then incubated with Alexa 555-UDF at 37 °C for 20 min. Samples were observed with an LSM710 confocal microscope. Images were captured with a 63× oil immersion objective and λex 543 nm. The emission fluorescence was filtered at 565 nm. The fluorescence intensities of samples were also measured with flowcytometry.
4
Measuring Cell-Bead Interactions via Microscopy
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Carboxylated silica beads (3 μm, Kisker Biotech) were prepared as described above except that ConA-coated beads were incubated with biotinylated BSA previously labeled with an Alexa Fluor 555 protein labeling kit (Invitrogen). Unlabeled beads (FN-coated) and labeled beads (ConA-coated) were mixed in the same proportion (1:1). Cells were allowed to spread for 15 min and then fixed with 4% PFA, permeabilized with 0.1% Triton-X 100, and incubated at room temperature for 1 hr with the indicated antibodies. Only beads at the cell periphery were analyzed (excluding cells in the ectoplasm-endoplasm border zone). To quantify fluorescence intensity, a 10-pixel-diameter ring was drawn around each selected bead using ImageJ. Mean fluorescence per area was normalized and plotted.
5
Immunostaining Antibody Protocols for Tissue Analysis
6
Multicolor Immunostaining with Diverse Antibodies
7
Fluorescent TGFβ Labeling and Cell-Surface Binding
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TGFβ was fluorescently labeled using the AlexaFluor-555 protein Labeling Kit (A20174, Invitrogen) according to manufacturer's instructions. For cell-surface binding of fluorescent TGFβ, cells were washed with HBSS at 4°C and incubated with A555-TGFβ (500ng/mL) in HBSS at 4°C for 30min. Cells were extensively washed and processed further for immunofluorescence.
8
Quantifying Fluorescent Bead Binding
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Carboxylated silica beads (3 μm, Kisker Biotech) were prepared as described above except that ConA-coated beads were incubated with biotinylated BSA previously labeled with an Alexa Fluor 555 protein labeling kit (Invitrogen). Unlabeled beads (FN-coated) and labeled beads (ConA-coated) were mixed in the same proportion (1:1). Cells were allowed to spread for 15 min and then fixed with 4% PFA, permeabilized with 0.1% Triton-X 100, and incubated at room temperature for 1 h with the indicated antibodies. Only beads at the cell periphery were analyzed (excluding cells in the ectoplasm-endoplasm border zone). To quantify fluorescence intensity, a 10-pixel-diameter ring was drawn around each selected bead using ImageJ. Mean fluorescence per area was normalized and plotted.
9
Coagulation Factor Binding Assay
10
Labeling of Recombinant IL-37 with Alexa Fluor 555
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To investigate the internalization of recombinant IL-37 into human AVICs and co-localization with other proteins in cells, IL-37 was labeled using the Alexa Fluor 555 Protein Labeling Kit (Thermo Fisher Scientific, Waltham, MA)) according to instructions provided by the manufacturer. In brief, 1 M sodium bicarbonate solution (pH 8.3) were added to 1 mg/mL solution of recombinant IL-37, followed by incubation with the reactive dye in the vial for 15 minutes at room temperature. To remove excess dye, the reaction mixture was added into a spin column contain resin. The final Alexa 555-labeled IL-37 was stored at -20 °C.
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