SH-SY5Y cells were seeded on glass coverslips and treated for 15 min with oligomers of various proteins at the listed concentrations in the absence or presence of SQ. After incubation, the cells were washed with PBS and counterstained with 5.0 μg/ml of Alexa Fluor 633-conjugated wheat germ agglutinin (Life Technologies, CA, United States) (Perni et al., 2017 (link)). After washing with PBS, the presence of oligomers was detected with 1:800 diluted mouse monoclonal 6E10 anti-Aβ antibodies (BioLegend, San Diego, CA, United States) or 1:800 rabbit anti-HypF-N antibodies (Primm, Milan, Italy) and subsequently with 1:1,000 diluted Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Life Technologies, CA, United States). Fluorescence emission was detected after double excitation at 488 and 633 nm by a scanning confocal microscopy system (Perni et al., 2017 (link)), and three apical sections were projected as a single composite image by superimposition. ImageJ (NIH, Bethesda, MD, United States) and JACOP plugin (rsb.info.nih.gov ) software were used to calculate the percentage of colocalization between cell membranes and oligomers.
Alexa fluor 633 conjugated wheat germ agglutinin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 633-conjugated wheat germ agglutinin is a fluorescent-labeled lectin used for labeling and detecting glycoconjugates in biological samples. It binds to N-acetylglucosamine and sialic acid residues on the cell surface.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal 6e10 anti aβ antibodies, by BioLegend (2 mentions)
Alexa fluor 488 conjugated anti mouse or anti rabbit secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Sodium bicarbonate buffer, by Merck Group (1 mentions)
Biocoat poly d lysin laminin, by Corning (1 mentions)
Oil immersion objective, by Nikon (1 mentions)
Glass coverslip, by Corning (1 mentions)
Alexa fluor 488 conjugated anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 protocols using alexa fluor 633 conjugated wheat germ agglutinin
1
Oligomer Localization and Cytotoxicity Assay in SH-SY5Y Cells
2
Trodusquemine Inhibits Amyloid Oligomer Binding
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SH-SY5Y cells were seeded on glass coverslips and treated for 15 min with HypF-N oligomers (6 µM) or Aβ40 oligomers (5 µM) or in the absence or presence of increasing concentrations of trodusquemine (10:1, 3:1, and 1:1 ratios of oligomers to trodusquemine, monomer equivalents). After incubation, the cells were washed with PBS and counterstained with 5.0 µg mL−1 Alexa Fluor 633-conjugated wheat germ agglutinin (Life Technologies, CA, USA)22 (link),28 (link). After washing with PBS, the presence of Aβ40 or HypF-N oligomers was detected with 1:800 diluted mouse monoclonal 6E10 anti-Aβ antibodies (BioLegend, CA, USA) or 1:800 diluted rabbit monoclonal anti-HypF-N antibodies (Primm, Milan, Italy) and subsequently with 1:1000 diluted Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Life Technologies, CA, USA) to recognize the 6E10 and anti-HypF-N primary antibodies, respectively. Fluorescence emission was detected after double excitation at 488 nm and 633 nm by the scanning confocal microscopy system described previously22 (link) and three apical sections were projected as a single composite image by superimposition. The percentages of oligomer colocalization were determined by analyzing regions of interest corresponding to 50–60 cells per condition.
3
Fluorescent Labeling and Imaging of Melittin
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To label
melittin, 300 μM Alexa Fluor 488 N-hydroxysuccinimide
(NHS) ester (succinimidyl ester, Invitrogen, ThermoFisher Scientific,
CA) was incubated with gentle shaking for 2 h with 900 μM melittin
in 0.1 mM sodium bicarbonate buffer (pH 8.0, Sigma-Aldrich, MO). SH-SY5Y
cells were seeded on glass coverslips (Corning BioCoat Poly-d -Lysin/Laminin, NY) and treated for 5 min with 0.2 μM labeled
melittin in the absence or presence of 0.1, 1.0, and 10 μM claramine.
After incubation, the cells were washed with phosphate-buffered saline
(PBS) and counterstained with 5 μg/mL Alexa Fluor 633-conjugated
wheat germ agglutinin (Life Technologies, CA).13 (link) After washing with PBS, cells were fixed in 2% paraformaldehyde.
Fluorescence emission was detected after double excitation at 488
and 633 nm by the above-described scanning confocal microscopy system
using a 60× oil immersion objective (Nikon Instruments). A series
of 1.0 μm thick optical sections (1024 × 1024) were acquired,
and all sections were projected as a single composite image by superimposition.
ImageJ (NIH, Bethesda, MD) was used to calculate the percentage of
colocalization between cell membranes and melittin.
melittin, 300 μM Alexa Fluor 488 N-hydroxysuccinimide
(NHS) ester (succinimidyl ester, Invitrogen, ThermoFisher Scientific,
CA) was incubated with gentle shaking for 2 h with 900 μM melittin
in 0.1 mM sodium bicarbonate buffer (pH 8.0, Sigma-Aldrich, MO). SH-SY5Y
cells were seeded on glass coverslips (Corning BioCoat Poly-
melittin in the absence or presence of 0.1, 1.0, and 10 μM claramine.
After incubation, the cells were washed with phosphate-buffered saline
(PBS) and counterstained with 5 μg/mL Alexa Fluor 633-conjugated
wheat germ agglutinin (Life Technologies, CA).13 (link) After washing with PBS, cells were fixed in 2% paraformaldehyde.
Fluorescence emission was detected after double excitation at 488
and 633 nm by the above-described scanning confocal microscopy system
using a 60× oil immersion objective (Nikon Instruments). A series
of 1.0 μm thick optical sections (1024 × 1024) were acquired,
and all sections were projected as a single composite image by superimposition.
ImageJ (NIH, Bethesda, MD) was used to calculate the percentage of
colocalization between cell membranes and melittin.
4
Visualization of α-Hemolysin Binding
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SH-SY5Y cells were seeded
on glass coverslips (Corning) and treated
for 15 min with 5 μg/mL (e.g., about 0.15 μM in monomer
equivalents) of α-hemolysin in the absence or presence of 0.1
and 10 μM claramine. After incubation, the cells were washed
with PBS, counterstained with 10 μg/ml Alexa Fluor 633-conjugated
wheat germ agglutinin (Life Technologies, CA),13 (link) and fixed in 2% paraformaldehyde. After washing with PBS,
the presence of α-hemolysin was detected with 1:750 diluted
rabbit antistaphylococcal α-toxin primary antibodies (Sigma-Aldrich,
MO) and subsequently with 1:1000 diluted Alexa Fluor 488-conjugated
antirabbit secondary antibodies (Life Technologies, CA). Fluorescence
emission was detected after double excitation at 488 and 633 nm by
the above-described scanning confocal microscopy system using a 20×
objective (Nikon Instruments). A series of 1.0 μm thick optical
sections (1024 × 1024) were acquired, and all sections were projected
as a single composite image by superimposition. ImageJ (NIH, Bethesda,
MD) was used to calculate the percentage of colocalization between
cell membranes and α-hemolysin.
on glass coverslips (Corning) and treated
for 15 min with 5 μg/mL (e.g., about 0.15 μM in monomer
equivalents) of α-hemolysin in the absence or presence of 0.1
and 10 μM claramine. After incubation, the cells were washed
with PBS, counterstained with 10 μg/ml Alexa Fluor 633-conjugated
wheat germ agglutinin (Life Technologies, CA),13 (link) and fixed in 2% paraformaldehyde. After washing with PBS,
the presence of α-hemolysin was detected with 1:750 diluted
rabbit antistaphylococcal α-toxin primary antibodies (Sigma-Aldrich,
MO) and subsequently with 1:1000 diluted Alexa Fluor 488-conjugated
antirabbit secondary antibodies (Life Technologies, CA). Fluorescence
emission was detected after double excitation at 488 and 633 nm by
the above-described scanning confocal microscopy system using a 20×
objective (Nikon Instruments). A series of 1.0 μm thick optical
sections (1024 × 1024) were acquired, and all sections were projected
as a single composite image by superimposition. ImageJ (NIH, Bethesda,
MD) was used to calculate the percentage of colocalization between
cell membranes and α-hemolysin.
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