Performance Comparison of Commercial Fixing and Permeabilizing Reagents

Abstract

Flow cytometry is commonly used to characterize cellular antigens expressed both on the cell surface and inside the cell. As the technology advances, more researchers are using this tool to study intracellular activity to understand how cells communicate and respond to their environment. Intracellular targets of interest include cytoplasmic antigens and proteins, nuclear antigens and proteins, cytokines, etc. The quality of intracellular staining depends on the ability of the permeabilizing reagent to permeabilize the cell membrane for antibodies to get to the target of interest without destroying the cell. Five different commercial permeabilizing reagents were evaluated in a small study, Caltag Fix and Perm® Kit, BD IntraSure™ Kit**This product is produced under license from Leeds Teaching Hospitals NHS Trust., BD FACS™ Permeabilizing Solution 2 (FACS Perm 2), 1X FACS Lysing Solution, and BD Cytofix/Cytoperm™ Kit. Five categories of performance were evaluated: cell recovery, maintenance of surface staining, preservation of light scattering properties, intracellular staining, and background staining. Cell recovery was assessed using CD45 APC and BD TruCOUNT™ tube; surface staining maintenance was assessed using CD45 APC, CD45 PerCP-Cy5.5, CD19 PE, and CD20 PerCP; intracellular and background staining were assessed using anti-bcl-2 FITC, anti-Zap 70 PE, anti-TdT FITC, anti-Ki-67 FITC, and CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5 reagents. Caltag and BD IntraSure Kits performed well in the category of cell recovery. Caltag Kit, BD IntraSure Kit, and FACS Lysing Solution maintained cell surface staining after cell permeabilization. BD IntraSure Kit and FACS Lysing Solution preserved light scattering properties of cells after permeabilization. Using signal-to-noise ratio as the criterion to evaluate background and intracellular positive staining, FACS Lysing Solution and FACS Perm 2 detected anti-bcl-2 FITC; BD IntraSure Kit and FACS Lysing Solution detected anti-TdT FITC; BD IntraSure Kit, FACS Lysing Solution, FACS Perm 2, and BD Cytofix/Cytoperm Kit detected anti-Ki-67 FITC; BD IntraSure Kit, FACS Lysing Solution, and FACS Perm 2 detected anti-Zap 70 PE; all kits detected CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5. Results showed that there is no universal permeabilizing reagent for all intracellular staining assays. Each test reagent has its own strength and weakness. Choosing the best permeabilizing reagent for a particular assay depends on the target of interest and the output of the assay.