293T cells were seeded at 20% density in 48-well plates at 12 to 15 h before transfection. Cells in each well were then transfected with 0.5 μl of Lipofectamine 2000 (Life Technologies, catalog no. 11668019) in complex with 200 ng of plasmid encoding one of the 16 ACE2 orthologs or a D30E mutant of the human ACE2. Culture medium was changed at 6 h after transfection. Cells were then detached with 5 mM EDTA (Life Technologies, catalog no. 15575020) at 36 h posttransfection. The cells were then stained with 5 μg/ml RBD-Ig proteins at 37°C for 10 min, washed three times, and then stained with 2 μg/ml Alexa488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, catalog no. A-11001) at room temperature for 20 min. After another three washes, cells were analyzed by Attune NxT flow cytometer (Thermo Fisher), and signals of 10,000 FSC/SSC-gated cells were collected for each sample.
Alexa 488 conjugated goat anti mouse igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody is a fluorescently labeled antibody that specifically binds to mouse immunoglobulin G (IgG) antibodies. The Alexa Fluor 488 dye provides a green fluorescent signal that can be detected using appropriate fluorescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Superblock t20 pbs blocking buffer, by Thermo Fisher Scientific (2 mentions)
Alexa 594 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Fluoview fv1000, by Olympus (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Wga fitc, by GeneTex (1 mentions)
Alexa 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti ve cadherin monoclonal antibody, by Beckman Coulter (1 mentions)
3 protocols using alexa 488 conjugated goat anti mouse igg secondary antibody
1
ACE2 Ortholog Binding Assay
2
Visualizing Endothelial Glycocalyx Integrity
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HUVECs were seeded as a monolayer onto a microscope cover glass slide and cultured under different conditions. After treatment for indicated time, the cells were fixed in 2% paraformaldehyde and then blocked with Superblock T20 (PBS) blocking buffer (Thermo Fisher Scientific).To measure the integrity of the endothelial glycocalyx and the deposition of CD138, the expression of sialic acid was stained with wheat germ agglutinin (WGA) lectin conjugated to FITC (WGA-FITC, Genetex) and the distribution of HPA-1 and CD138 was detected by anti-mouse-CD138 mAb (BD, Franklin Lakes, NJ, USA) or rabbit anti-HPA-1 polyclonal antibody (GeneTex). Primary antibodies were incubated with the fixed monolayer overnight at 4°C, followed by incubation with Alexa 488-conjugated goat anti-mouse IgG secondary antibody, Alexa 594-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, CA, USA) (1:500 diluted) and Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) (1:3,000 diluted) for 1 h. Images were captured using a confocal microscope (Olympus FluoView FV1000, Melville, NY, USA).
3
Immunofluorescence Analysis of Cell-Cell Junctions and Signaling Receptors
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A monolayer of cells was seeded onto a microscope cover glass slide. After 30 min of treatment, the cells were fixed in 4% paraformaldehyde for 15 min followed by three washes with PBS. The cells were subsequently blocked with Superblock T20 (PBS) Blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. To detect VE-cadherin, LC3 puncta localization, or the expression of the MIF receptors, a mouse anti-VE-cadherin monoclonal antibody (Beckman Coulter), a rabbit anti-LC3 polyclonal antibody (GeneTex), a goat anti-CD74 polyclonal antibody (SantaCruz), a rabbit anti-CXCR4 polyclonal antibody (GeneTex), or a rabbit anti-CXCR7 (GeneTex) polyclonal antibody (1:200 diluted in PBS) were incubated with the cells overnight at 4°C. After three washes with Tris-buffered saline-Tween 20 (TBST), the cells were incubated with an Alexa 488-conjugated goat anti-mouse IgG secondary antibody, an Alexa 594-conjugated goat anti-rabbit IgG secondary antibody or an Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, Calif) (1:500 diluted) and Hoechst 33342 (Invitrogen) (1:3,000 diluted) for 1 h, and the slides were washed 3 times with PBS. The images were acquired using a confocal microscope (Olympus FluoView FV1000, Melville, NY).
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