The flow cytometry was carried out using the BD FACSCanto™ II Cell Analyzer (BD Bioscience). The analysis of THP-1 macrophages, human classical monocytes, and Kupffer cells was performed as described previously with some modifications (Tian et al., 2016 (link)). Briefly, cells were collected and incubated with primary antibodies IgG2a-PE, IgG1-APC, CD14-PE, APC anti-human CD11b antibody (Biolegend, #301310), APC anti-human CD16 antibody (Biolegend, #360705), F4/80-PE (eBioscience, #12-4801-82), or IgG2a-PE (eBioscience, #12-4321-80) at 4°C for 30 minutes. Cells were rinsed with PBS three times before the flow cytometry analysis. Data were analyzed by FlowJo 10 software (BD Bioscience)
Igg2a pe
Manufactured by Thermo Fisher Scientific
IgG2a-PE is a fluorescently-labeled antibody used for flow cytometry applications. It is a murine immunoglobulin G2a subclass conjugated to the phycoerythrin (PE) fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 cell analyzer, by BD (2 mentions)
F4 80 pe, by Thermo Fisher Scientific (2 mentions)
Cd14 pe, by BioLegend (2 mentions)
Igg2a pe, by BioLegend (2 mentions)
Anti human cd11b apc antibody, by BioLegend (2 mentions)
Apc anti human cd16 antibody, by BioLegend (2 mentions)
Igg1 apc, by BioLegend (2 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cd3 pe, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Cd43 pe cy7, by BD (1 mentions)
70 m cell strainer, by BD (1 mentions)
Igg1 pe cy7, by BD (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Cd4 allophycocyanin, by BioLegend (1 mentions)
Igd pe, by BioLegend (1 mentions)
Gl7 af488, by BioLegend (1 mentions)
4 protocols using igg2a pe
1
Flow Cytometry Analysis of Macrophages
2
Flow Cytometry Analysis of Macrophages
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The flow cytometry was carried out using the BD FACSCanto™ II Cell Analyzer (BD Bioscience). The analysis of THP-1 macrophages, human classical monocytes, and Kupffer cells was performed as described previously with some modifications (Tian et al., 2016 (link)). Briefly, cells were collected and incubated with primary antibodies IgG2a-PE, IgG1-APC, CD14-PE, APC anti-human CD11b antibody (Biolegend, #301310), APC anti-human CD16 antibody (Biolegend, #360705), F4/80-PE (eBioscience, #12-4801-82), or IgG2a-PE (eBioscience, #12-4321-80) at 4°C for 30 minutes. Cells were rinsed with PBS three times before the flow cytometry analysis. Data were analyzed by FlowJo 10 software (BD Bioscience)
3
Multicolor Flow Cytometry of Murine Hematopoietic Cells
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Single-cell suspension of bone marrow was obtained by mechanical dissociation using mortar and pestle, and whole spleens were mashed into suspension using a 70-µm cell strainer (BD Falcon). Bone marrow cells, splenocytes, and peripheral blood from mice were red cell-lysed in ACK lysis buffer, and cells were incubated with cell surface FACS antibodies (in FACS buffer; 2% FCS in PBS) for at least 1 h on ice before analysis on a BD LSR II or BD LSR Fortessa flow cytometer. For staining with antibody cocktails, compensation was set using single-stained controls. B220-APCCy7 (103223), B220-APC (103211), and Flt3-PE (135306) were purchased from Biolegend. CD3-PE (561799), Mac1-PerCPCy5.5 (550993), IgD-PE (558597), IgM-APC (550676), CD19-PECy7 (552854), CD24-PerCPCy5.5 (562360), and CD43-PECy7 (562866) were purchased from BD Biosciences. ckit-PerCP eFluor710 (46-1171-80), hTSLPR (CRLF2)-PE (12-5499-42), and hCD127-PECy7 (25-1278-41) were purchased from eBioscience. mTSLPR (Crlf2)-PE (FAB5461P) was purchased from R&D Systems. An IgG isotype antibody was used as a negative control for IL7R (IgG1-PECy7; BD Biosciences, 557872) and CRLF2 (IgG2a-PE; eBioscience, 12-4724-82).
4
Immunohistochemical Analysis of Splenic and Renal Tissues
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Splenic and kidney sections were embedded in Tissue-Tek OCT compound (Sakura Finetek) and rapidly frozen in a freezing bath of dry ice and 2-methylbutane. Frozen OCT samples were cryosectioned and unstained slides were stored at −80°C. Immunohistochemical staining procedures were performed as previously described (1 (link), 2 (link), 5 (link)). For detection of germinal centers in the spleen, the following monoclonal Abs were used: GL7-AF488, IgD-PE, and CD4-allophycocyanin (all from BioLegend). For detection of renal deposition, C3-FITC (Cedarlane, Burlington, NC) and IgG2a-PE (eBioscience) Abs were used. Images were captured with a Zeiss LSM 880 confocal microscope (Fralin Imaging Center, Virginia Tech). Corrected total cell fluorescence scores were calculated with ImageJ software (National Institutes of Health, Rockville, MD).
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