Flow Cytometry FAQs

This product cannot be used for column-based sorting; it can only be used for tube-based sorting with a magnetic stand.

Storage Conditions: Store antibodies at 2–8°C, protected from light. Do not freeze unless specifically instructed. Both fluorochromes and antibody proteins are highly sensitive to freeze-thaw cycles; repeated freezing and thawing can lead to protein denaturation, fluorescence quenching, or aggregate formation. For long-term storage, consider aliquoting into smaller volumes to avoid repeat handling. Transport Effects: Short-term ambient temperature transport generally does not significantly compromise antibody titer or performance. However, if stored at room temperature for longer than one week, antibody efficacy may decline. Handling Recommendation: Upon receipt, briefly centrifuge the vial to collect any liquid from the cap and walls into the bottom of the tube to prevent loss during pipetting.

Importance of Titration: More antibody does not necessarily yield better results. Excessively high concentrations increase non-specific binding and background noise, while insufficient concentrations result in weak signals or false negatives. It is essential to perform an antibody titration prior to formal experiments to determine the optimal working concentration—the point at which antigen saturation is achieved with minimal background. Format Differences: “Test-size” antibodies are typically pre-titrated by the manufacturer and can be used according to the datasheet (e.g., 5 µL per 100 µL staining volume). For bulk (µg) formats, the optimal concentration must be determined empirically. Cell Number Adjustments: Antibody volume is primarily dictated by the total staining volume rather than the absolute cell count. If the cell number is reduced but the staining volume remains at 100 µL, the antibody volume usually remains unchanged. To conserve reagents, both cell numbers and antibody volumes should be scaled down proportionally; however, note that very low cell counts increase losses during centrifugation and washing steps.

Isotype Control: Used to assess background fluorescence resulting from Fc receptor binding or non-specific interactions. The isotype control must match the primary antibody’s species, isotype, fluorochrome, and concentration exactly. Fluorescence Minus One (FMO) Control: Critical for defining positive vs. negative population boundaries in multi-color panels, particularly when gating is ambiguous. Single-Stained Control: Essential for setting compensation matrices in multi-color flow cytometry. If the sample lacks strong positive populations, compensation beads can be used as substitutes. Selection Guidance: Beginners troubleshooting background issues may start with isotype controls; for precise gating in complex panels, FMOs are preferred; single-stained tubes (or bead controls) are mandatory for compensation setup.

Direct Conjugation: Fluorochromes are directly coupled to the primary antibody. This method involves fewer steps, yields lower background, and offers superior reproducibility, making it the gold standard for flow cytometry. Indirect Staining: Utilizes a primary antibody followed by a fluorescently labeled secondary antibody. While this amplifies signal sensitivity, it significantly increases the risk of non-specific binding and high background due to cross-reactivity. Selection Guidance: Direct conjugates are strongly recommended for routine surface staining. Indirect methods should only be considered when no direct conjugate is available or when target antigen expression is extremely low; rigorous controls (secondary-only or isotype controls) are mandatory in such cases.