The following anti-human monoclonal antibodies (clones) from eBiosciences, San Diego CA, were used for surface staining of PBMCs for total T cells, subsets and activation: CD3 (HIT3a), CD4 (SK3), CD8 (SK1) and CD69 (FN50) and Ki-67 (20Raj1) was used for intracellular staining to assess turnover. Intracellular expression of OPG and RANKL in T cells, was performed as previously described [10 (link)]. Briefly, cells were first stained for surface markers and then fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/permeabliziation kit (BD Biosciences). To quantify intracellular OPG, cells were incubated with 5 μg/ml of anti-human OPG-biotin (Leinco Technologies) for 30 minutes at 4°C,followed by incubation with Streptavidin-PE. RANKL was quantified by incubation with 5 μg/ml recombinant OPG-Fc (R&D systems) for 30 minutes at 4°C as previously described [12 (link)], followed by anti-human IgG Fc-PE (BD Biosciences) for another 30 minutes at 4°C. All cells were fixed in 4% paraformaldehyde, data acquired on an Accuri flow cytometer (BD Immunocytometry Systems, San Jose, CA) and analyzed using FlowJo software version 9.7 (Ashland, OR).
Anti human igg fc pe
Manufactured by BD
Anti-human IgG Fc-PE is a lab equipment product that detects human immunoglobulin G (IgG) antibodies. It contains phycoerythrin (PE) as a fluorescent label, which allows for the identification and quantification of IgG antibodies in samples.
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2 protocols using anti human igg fc pe
1
Quantifying T Cell Activation Markers
2
PBMC Intracellular Staining for OPG and RANKL
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Thawed PBMC were rested overnight at 37°C, and cells were incubated with a protein transport inhibitor containing monensin (Golgistop, BD Biosciences) for the last 5 hours of the 16- to 18-hour recovery period. For intracellular staining cells were first stained for surface markers and then fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/permeabliziation kit (BD Biosciences). To quantify intracellular OPG, cells were incubated with 5 µg/ml of anti-human OPG-biotin (Leinco Technologies) for 30 minutes at 4°C, followed by incubation with Streptavidin-PE. RANKL was quantified by incubation with 5 µg/ml recombinant OPG-Fc (R&D systems) for 30 minutes at 4°C as previously described [43] (link), followed by anti-human IgG Fc-PE (BD Biosciences) for another 30 minutes at 4°C. Gates were set as shown in Supplemental Figure S1 , using fluorescence-minus-one (FMO) controls to establish specific binding ranges. All cells were fixed in 4% paraformaldehyde and analyzed on an Accuri flow cytometer (BD Immunocytometry Systems, San Jose, CA). Data were analyzed using FlowJo software version 9.6 (Treestar, San Carlos CA).
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