To screen the human surface marker of MSCs, 242 antibodies were lyophilized in 96-well plates (BD LysoplatesTM; BD Biosciences) at 0.5 μg/well and incubated with 500,000 MSCs per well. With 20 min reconstitution on ice, the washed cells were stained with an Alexa Fluor® 647-conjugated goat-anti-mouse IgG secondary antibody (Molecular Probes, Eugene, OR). Flow cytometry was performed to measure the surface markers using a FACSCalibur instrument (BD Biosciences). The data from flow cytometry were analyzed in Excel 2013 (Microsoft, Redmond, WA) to generate heat maps [20 (link)].
Alexafluor 647 conjugated goat anti mouse igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
AlexaFluor 647-conjugated goat anti-mouse IgG secondary antibody is a fluorescently-labeled detection reagent used to identify and visualize mouse primary antibodies in various immunoassay applications.
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Lab products found in correlation
Facscalibur, by BD (3 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Normal goat igg, by Santa Cruz Biotechnology (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Cathepsin d, by Santa Cruz Biotechnology (1 mentions)
Vps34, by Cell Signaling Technology (1 mentions)
Gαq 11, by Santa Cruz Biotechnology (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
Beclin 1, by Santa Cruz Biotechnology (1 mentions)
Liberase research grade, by Roche (1 mentions)
Medium 199, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Igfbp 3 antibody, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using alexafluor 647 conjugated goat anti mouse igg secondary antibody
1
Screening MSC Surface Markers
2
Screening Surface Markers of hMSCs
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To screen the surface markers of hMSCs, 242 antibodies (Figure S1 ) were lyophilized in 96-well plates (BD Lyoplates; BD Biosciences) at 0.5 µg/well and incubated with 500,000 MSCs per well. After 20 min of reconstitution on ice, the washed cells were stained with an Alexa Fluor® 647-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes, Eugene, OR, USA). Flow cytometry was performed to confirm the surface marker expression on a FACSCalibur instrument (BD Biosciences). Flow cytometry data were analyzed using Excel 2013 (Microsoft, Redmond, WA, USA) to generate heat maps [28 (link)].
3
Phenotypic Characterization of Kidney-Derived Stem Cells
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P3 KSCs were suspended in 100 μL PBS buffer pH 7.4 and labeled with allophycocyanin (APC)-conjugated monoclonal rat CD29 antibody (Biolegend, USA), phycoerythrin (PE)-conjugated monoclonal CD90 antibody (BD, USA), or peridinin chlorophyll protein (PerCP)-conjugated monoclonal CD45 antibody against (Biolegend, USA) for 30 min at 4°C. For CD73 immunostaining, KSCs were incubated with 2 μL mouse anti-rat CD73 antibody (BD, USA) at 4°C on a shaker for 30 min, followed by AlexaFluor 647-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, USA). Cells were washed three times in cold PBS buffer and then analyzed on a BD FACSCalibur (BD Biosciences, Canada).
4
Antibody Sourcing and Specialty Chemical Protocol
5
Immunofluorescence Assay for IGFBP-3
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Cells were fixed with 4% paraformaldehyde in PBS for 15 min. After washing with PBS, cells were permeabilized by incubating in PBS-T for another 15 min. After blocking with 10% normal goat serum (Thermo Fisher Scientific) for 30 min, cells were incubated for 1 h with IGFBP-3 antibody (Santa Cruz Biotechnology). Next, cells were washed PBS-T before being incubated with Alexa Fluor 647-conjugated goat anti–mouse IgG secondary antibody (Invitrogen) and Hoechst 33342 (Invitrogen) for 30 min. Cells were subsequently imaged with a fluorescence microscope.
6
Visualizing SARS-CoV-2 Spike Protein and Dendritic Cells
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Local skin tissues from immunized mice were immediately frozen in liquid nitrogen. The tissue sections were embedded, sliced, fixed, and blocked using 5% bovine serum albumin (BSA). For detection of the viral antigen, the sections were sequentially incubated with a primary mouse anti-SARS-CoV-2 spike antibody (SinoBiological Co., Ltd) and an AlexaFluor 647-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA, USA). Dendritic cells were detected using anti-CD11c antibody (Abcam, Cambridge, UK) and Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (Invitrogen). The cell nuclei were detected using DAPI. Fluorescence was visualized and analyzed using a confocal microscope (TCS SP2, Leica).
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