Phagocytic assays were performed using the pHrodo Green E. coli Bioparticle conjugate system (cat# P35366, Invitrogen) according to manufacturer recommendations. Activation of fluorescence of the pHrodo Green in this system is pH dependent and is detected only when internalized within the phagosome of the cell. Green E. coli Bioparticle conjugate was reconstituted in PBS, diluted in media, and incubated with the cells for 1 hour at 37C. Cells were then washed with cold PBS three times to reduce non-specific attachment collected using a cell scraper. Collected macrophages were then treated with Fc Receptor blocker for 10 minutes and stained for 20 minutes at 4° C with PerCP 5.5-CD14 and with APC-CD90 (5E10, cat # 328113, Biolegend) for gating of MEMs. CD14 positive/pH pHrodo Green positive cells at Ex/Em of 509/533 nm were detected on the MACSQuant analyzer 10 (Miltenyi Biotec) and analyzed using FlowJo software (FlowJo, Inc, Ashland, OR).
Apc cd90 5e10
Manufactured by BioLegend
APC-CD90 (5E10) is a fluorescent antibody that binds to the CD90 (Thy-1) antigen. CD90 is a glycophosphatidylinositol (GPI)-anchored protein expressed on the surface of various cell types, including T cells, hematopoietic stem cells, and certain neurons. This antibody is conjugated with the fluorescent dye Allophycocyanin (APC), which can be detected using flow cytometry.
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Lab products found in correlation
2 protocols using apc cd90 5e10
1
Phagocytic Assay with E. coli Bioparticles
2
Multiparametric Flow Cytometry Characterization
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Cells were co-labeled with propidium iodide, to gate on viable populations. During data analysis, MSCs were excluded from MDMs by CD90 positive cell exclusion, and from Th cells by CD4 negative cell exclusion. Cells were characterized using a FC500 flow cytometer and analyzed on FlowJo (Treestar). Antibodies used were: FITC-CD4 (OKT4), PE-Cy7-CD14 (61D3), PE-CD206 (19.2), PE-CD127 (eBioRDR5) and PE-Cy7-CD25 (BC96) (eBioscience); FITC-CD163 (GHI/61) and APC-CD90 (5E10) (Biolegend, San Diego, CA).
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