title: Polystyrene microspheres enable 10-color compensation for immunophenotyping of primary human leukocytes.
abstract: Compensation is a critical process for the unbiased analysis of flow cytometry data. Numerous compensation strategies exist, including the use of bead-based products. The purpose of this study was to determine whether beads, specifically polystyrene microspheres (PSMS) compare to the use of primary leukocytes for single color based compensation when conducting polychromatic flow cytometry. To do so, we stained individual tubes of both PSMS and leukocytes with panel specific antibodies conjugated to fluorochromes corresponding to fluorescent channels FL1-FL10. We compared the matrix generated by PSMS to that generated using peripheral blood mononuclear cells (PBMC). Ideal for compensation is a sample with both a discrete negative population and a bright positive population. We demonstrate that PSMS display autofluorescence properties similar to PBMC. When comparing PSMS to PBMC for compensation PSMS yielded more evenly distributed and discrete negative and positive populations to use for compensation. We analyzed three donors' PBMC stained with our 10-color T cell subpopulation panel using compensation generated by PSMS vs.PBMC and detected no significant differences in the population distribution. Panel specific antibodies bound to PSMS represent an invaluable valid tool to generate suitable compensation matrices especially when sample material is limited and/or the sample requires analysis of dynamically modulated or rare events.
authors.affiliation: Translational Biology and Molecular Medicine Graduate Program, Baylor College of Medicine, Houston, Texas, 77030.|Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, 77030.|Cancer Center, Texas Children's Hospital, Houston, Texas, 77030.
authors.forename: Tiara
authors.initials: T
authors.lastname: Byrd
authors.affiliation: Beckman Coulter Inc, Brea, California.
authors.forename: Karen D
authors.initials: KD
authors.lastname: Carr
authors.affiliation: Beckman Coulter Inc, Brea, California.
authors.forename: John C
authors.initials: JC
authors.lastname: Norman
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, 77030.|Cancer Center, Texas Children's Hospital, Houston, Texas, 77030.
authors.forename: Leslie
authors.initials: L
authors.lastname: Huye
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, 77030.|Cancer Center, Texas Children's Hospital, Houston, Texas, 77030.
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Hegde
authors.affiliation: Translational Biology and Molecular Medicine Graduate Program, Baylor College of Medicine, Houston, Texas, 77030.|Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, 77030.|Cancer Center, Texas Children's Hospital, Houston, Texas, 77030.
authors.forename: Nabil
authors.initials: N
authors.lastname: Ahmed
mesh_terms: D000906:Antibodies / Q000378:metabolism; D003116:Color; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D007962:Leukocytes / Q000166:cytology / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008863:Microspheres; D011137:Polystyrenes / Q000276:immunology
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D011137:Polystyrenes
keywords: beads; compensation; flow cytometry; immunophenotyping; leukocytes; polychromatic; polystyrene microspheres