Flow cytometry was used to analyze binding of rabbit antibodies to spleen leukocytes [9 (link)]. The cells in PBS containing 1% BSA were blocked with 10% (v/v) donkey serum (Southern Biotech, Birmingham, AL) before or after incubation with rabbit polyclonal anti-mutated peptide antibodies (1 × 107 cells/1.1 μg each Ab/100μL). Cell-bound rabbit Ig was detected with phycoerythrin-conjugated donkey anti-rabbit Ig (H + L) antibody (BioLegend, San Diego, CA). All the incubation steps were for 30 min in ice followed by three-time (3 min each) washings with PBS containing 1% BSA. The stained cells were fixed in 2% paraformaldehyde and following PBS washes, the samples were run on BD LSRII (BD Biosciences, San Jose, CA) flow cytometer equipped with BD FACSDiva™ version 8 software for data acquisition. The data were analyzed using FlowJo™ v10.1 software (FlowJo, LLC, Ashland, OR) following the required gating with the help of proper controls.
Protocol detail
Flow Cytometry Analysis of Antibody Binding
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Anti antibodies
Antibodies
Antibody
Cells
Donkey
Flow cytometry
Leukocytes
Paraformaldehyde
Peptide
Phycoerythrin
Rabbit
Serum
Spleen
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Variable analysis
independent variables
- Incubation with rabbit polyclonal anti-mutated peptide antibodies (1 × 10^7 cells/1.1 μg each Ab/100μL)
- Binding of rabbit antibodies to spleen leukocytes
- Cells in PBS containing 1% BSA
- Blocking with 10% (v/v) donkey serum (Southern Biotech, Birmingham, AL) before or after incubation with rabbit polyclonal anti-mutated peptide antibodies
- Detection of cell-bound rabbit Ig with phycoerythrin-conjugated donkey anti-rabbit Ig (H + L) antibody (BioLegend, San Diego, CA)
- Incubation steps for 30 min in ice followed by three-time (3 min each) washings with PBS containing 1% BSA
- Fixing the stained cells in 2% paraformaldehyde and following PBS washes
- Samples run on BD LSRII (BD Biosciences, San Jose, CA) flow cytometer equipped with BD FACSDiva™ version 8 software for data acquisition
- Data analyzed using FlowJo™ v10.1 software (FlowJo, LLC, Ashland, OR) following the required gating with the help of proper controls
- None specified
- None specified
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