Computational protocol: Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency

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Protocol publication

[…] A whole blood staining protocol was used for the peripheral blood T cell immunophenotyping. Four hundred fifty milliliters of EDTA blood was labeled with anti–CD3-PE.Cy5, anti–CD4-BV711, anti–CD8a-allophycocyanin, anti–CD45RA-BV510, anti–CD27-BV605, anti–CD28-PE/Cy7, anti–CCR7-PE/Cy5.5, and anti-CD25-allophycocyanin/Cy7 Abs (Biolegend) to determine various T cell subpopulations. Abs were used in 1:100 dilutions and incubated at room temperature for 20 min in the dark. All cells were washed with isotonic PBS with 2% FCS and incubated with 2 ml of 0.16 M of NH4CL at room temperature in the dark for 10 min for red cell lysis. A final wash with PBS and 2% FCS was carried out, and 0.5 ml of 1% paraformaldehyde was added. CountBright Absolute Counting Beads (Life Technologies) were added according to manufacturer’s protocol for accurate cell counting. Compensation was aided by the Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD Biosciences) and automatically calculated by BD FACSDiva software version 6.0. Acquisition was performed via BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed via FlowJo version 7.6.5 software. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Homo sapiens
Diseases Deficiency Diseases, Immunologic Deficiency Syndromes, Infection, Common Variable Immunodeficiency