Single B cell sorting was followed as previously described (3 (link), 71 (link)). Briefly, PBMCs from donor CBJC438 were incubated with an antibody cocktail consisting of CD19-PE-Cy7, CD8-PerCP, IgM-PE-Cy5, IgG-FITC (all from BD Biosciences), CD20-ECD (Beckman), CD3–Pacific Orange (Invitrogen), CD14–eFluor 450 (eBioscience), BG505-APC (purified in laboratory), and LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) to exclude dead cells. The PBMCs were first stained with 50 μl of phosphate-buffered saline (PBS) with LIVE/DEAD at 4°C in the dark for 30 min and then stained with 50 μl of PBS with an antibody cocktail for 1 hour at 4°C in the dark. The stained cells were washed and resuspended in PBS and then passed through a 70-μm cell mesh (BD Biosciences). Antigen-specific single B cells were gated as CD19+CD20+CD8−CD3−CD14−IgM−IgG+BG505+ and sorted into a 96-well PCR plate containing 20 μl of lysis buffer: 5 μl of 5× first-strand buffer, 0.5 μl of RNaseOUT, 1.25 μl of 0.1 M dithiothreitol (all from Invitrogen), and 0.0625 μl of IGEPAL (Sigma-Aldrich) per well. The sorted plate was snap-frozen on dry ice and stored at −80°C before reverse transcription (RT) reaction.
Cd14 efluor450
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD14-eFluor450 is a fluorochrome-conjugated antibody that binds to the CD14 surface marker. CD14 is a glycosylphosphatidylinositol-anchored protein that is expressed on the surface of monocytes, macrophages, and neutrophils, and serves as a co-receptor for the detection of lipopolysaccharide. The eFluor450 fluorochrome enables detection of the CD14 antigen using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2 system, by BD (2 mentions)
Cd68 fluorescein isothiocyanate, by Thermo Fisher Scientific (2 mentions)
Ki67 allophycocyanin, by BioLegend (2 mentions)
Il 32 alexa fluor 488, by R&D Systems (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Igm pe cy5, by BD (1 mentions)
Igg fitc, by BD (1 mentions)
Igepal, by Merck Group (1 mentions)
Cd20 ecd, by Beckman Coulter (1 mentions)
Cd3 pacific orange, by Thermo Fisher Scientific (1 mentions)
70 μm cell mesh, by BD (1 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd8 percp, by BD (1 mentions)
Cd19 pe cy7, by BD (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Cd3 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd19 alexa fluor 647, by BioLegend (1 mentions)
Cd56 pe cy7, by Thermo Fisher Scientific (1 mentions)
5 protocols using cd14 efluor450
1
Single B Cell Sorting for Antigen-Specific Isolation
2
PBMC Processing and CD4+ T Cell Enrichment
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PBMC sample processing was performed at the same time for each triplet (PwMS at VIS1&VIS2 and HC), to minimize differences within processing. The MojoSort™ Human CD4 T Cell Isolation Kit (#480010, BioLegend, San Diego, CA, USA) was used for CD4+ T cell enrichment, in accordance with the manufacturer’s instructions.
To assess whether CD4+ T cell enrichment was achieved, flow cytometry was performed in a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) on a subset of samples (before and after magnetic enrichment). The following antibodies were used: CD14-eFluor450 (#48014941, clone 61D3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD19-Alexa Fluor 647 (#302222, clone HIB19, BioLegend, San Diego, CA, USA); CD3-PerCP-Cy5.5 (#45003741, clone OKT3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD4-BV711 (#317439, clone OKT4, BioLegend, San Diego, CA, USA); and CD56-PE-Cy7 (#25056741, clone CMSSB/NCAM, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA).
Regarding the gating strategy, CD4+ T cells were defined as CD3+CD4+CD19-CD56- single cells (see Supplementary Figs.S6 , 7 online). A detailed protocol and additional details regarding the methodology used for CD4+ T cell enrichment and flow cytometry may be found in Sect. 2 of the Supplementary Information.
To assess whether CD4+ T cell enrichment was achieved, flow cytometry was performed in a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) on a subset of samples (before and after magnetic enrichment). The following antibodies were used: CD14-eFluor450 (#48014941, clone 61D3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD19-Alexa Fluor 647 (#302222, clone HIB19, BioLegend, San Diego, CA, USA); CD3-PerCP-Cy5.5 (#45003741, clone OKT3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD4-BV711 (#317439, clone OKT4, BioLegend, San Diego, CA, USA); and CD56-PE-Cy7 (#25056741, clone CMSSB/NCAM, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA).
Regarding the gating strategy, CD4+ T cells were defined as CD3+CD4+CD19-CD56- single cells (see Supplementary Figs.
3
Flow Cytometry Immunophenotyping Protocol
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The flow cytometry antibodies CD14-eFluor 450 and CD68-fluorescein isothiocyanate (FITC) were purchased from eBioscience, Ki67-allophycocyanin (APC) was purchased from BioLegend, and IL-32–Alexa Fluor 488 was purchased from R&D Systems. An FITC annexin V apoptosis detection kit purchased from BD was used to detect the cell apoptosis fraction. Mononuclear cells isolated from TPE or macrophages differentiated from THP-1 cells were stained for flow cytometry as previously described (38 (link)) to analyze the expression of surface markers, intracellular markers, apoptosis, and proliferation incorporation using the three-laser FACSAria II system (BD Biosciences, USA). Marker staining was performed according to the manufacturer’s protocol.
4
Flow Cytometry Immunophenotyping Protocol
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The flow cytometry antibodies CD14-eFluor 450 and CD68-fluorescein isothiocyanate (FITC) were purchased from eBioscience, Ki67-allophycocyanin (APC) was purchased from BioLegend, and IL-32–Alexa Fluor 488 was purchased from R&D Systems. An FITC annexin V apoptosis detection kit purchased from BD was used to detect the cell apoptosis fraction. Mononuclear cells isolated from TPE or macrophages differentiated from THP-1 cells were stained for flow cytometry as previously described (38 (link)) to analyze the expression of surface markers, intracellular markers, apoptosis, and proliferation incorporation using the three-laser FACSAria II system (BD Biosciences, USA). Marker staining was performed according to the manufacturer’s protocol.
5
HIV-1 infection of monocyte-derived macrophages
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MDMs infected with HIV-1BaL-iGFP were lifted with 12 mM lidocaine/5 mM EDTA in PBS and added to DiD (Molecular Probes)-labeled HFAs seeded at 5 × 106 cells/dish. Cells were cocultured for 24 hr before lifting and fixation for 15 min in 4% formaldehyde in PBS. Cells were permeabilized in 0.1% saponin (Sigma) and 30% FCS for 30 min at RT before labeling for CD14 (CD14-eFluor450, eBioscience). Cells were analyzed on an ImageStream X Mark II (Amnis) with a 20× or 40× objective with extended depth of field (EDF) and data processed using Ideas v.6 (Amnis).
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