Flow Cytometry Staining Protocols for High-Quality Data

INTRANUCLEAR STAINING BIOTIN-STREPTAVIDIN SYSTEMS INDIRECT CELL SURFACE STAINING INTRACELLULAR STAINING GENTLE SAMPLE HANDLING • Centrifuge at minimum required speed (300–400g) • Keep samples at 4 °C throughout procedure • Avoid vigorous vortexing or bubble formation • Never leave cell pellets dry CLUMP PREVENTION • Add DNase I to buffers • Include EDTA to prevent calcium-dependent aggregation • Filter through 70 μm strainer before acquisition • Use HBSS without Ca²⁺/Mg²⁺ for neutrophil-rich samples TUBE SELECTION STRATEGY • Polypropylene tubes reduce monocyte adherence • Round-bottom tubes facilitate complete resuspension • Check cytometer compatibility before starting High background staining Low positive signal Compensation issues Low cell viability Cell clumping PROBLEM Insufficient washing, antibody concentration too high Antibody concentration too low, inadequate permeabilization for intracellular or intranuclear targets Inadequate single-color controls Harsh processing, extended room temperature storage DNA release from dead cells, Ca²⁺/Mg²⁺ in buffers LIKELY CAUSE Single cell suspension achieved Cell viability >85% No debris or clumps Appropriate cell concentration (10⁵–10⁷ cells/mL) CRITICAL SAMPLE QUALITY INDICATORS Sample Preparation Excellence Increase wash steps, titrate antibodies Optimize antibody concentration, extend permeabilization Use compensation beads if there are few positive cells Gentle handling, keep samples cold Add DNase I, use metal-free buffers SOLUTION All fluorescent conjugates should be kept in the dark to prevent photobleaching Include monensin or brefeldin A to ensure that cytokines remain internalized and are not released Use species-specific secondaries to avoid cross-reactivity. Include secondary-only controls to assess background binding ! TIP ! TIP ! TIP ! TIP ! TIP Flow Cytometry Staining Protocols: Shining a Light on High-Quality Data Five Essential Staining Approaches Different permeabilization buffers may be required depending on the antigen location Select the ideal fluorophore for your assay from a range of streptavidin formats DIRECT CELL SURFACE STAINING Recommended Best Practices Instruments and Reagents Educational materials Technical support Bio-Rad Solutions for Flow Cytometry Excellence Troubleshooting Quick Reference Thaw cells rapidly and immediately add to cold media or staining buffer containing bovine serum albumin to dilute out the cryoprotectant Always remove contaminating material during extraction (e.g., muscle tissue when flushing bone marrow) Choose anticoagulants carefully: avoid EDTA when detecting intracellular cytokines or Ca²⁺-dependent surface markers like integrins Use gentler dissociation methods than standard passaging protocols. Trypsin creates debris and destroys epitopes, so consider using enzyme-free or milder enzymatic alternatives Flow Cytometry Hub StarBrightTM Dyes TrailBlazerTM Kits ZE5 Cell Analyzer Flow Cytometry Reagents Bio-Rad and StarBright are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. TrailBlazer is a trademark of Bio-Rad Europe GmbH in certain jurisdictions. All trademarks used herein are the property of their respective owner. © 2025 Bio-Rad Laboratories, Inc. Additional Resources Staining challenges can leave you in the dark; whether its inconsistent results, protocol failures or troubleshooting delays. Let Bio-Rad’s expertise, educational materials, and high-performance products shed light on your flow cytometry experiment needs.