Suitable for Flow Cytometry

发布于 2025年10月24日更新时间 2025年10月24日

Flow cytometry analyzes cells at the single-cell level and, with multi-laser/multi-channel fluorescence detection, can rapidly measure high-dimensional cell phenotypes, functions, and microenvironments. The spread of multicolor and spectral flow makes the system highly sensitive to buffer composition, blocking strategies, fluorophore compatibility, fixation/permeabilization, and the quality of compensation/deconvolution. Reagents optimized and validated for flow applications can establish a stable balance among signal intensity, background suppression, and spectral separation, thereby improving reproducibility and cross-platform comparability.

I. Definition and Features

“Reagents suitable for flow cytometry” are formulation systems developed and validated around the flow staining and detection workflow, covering staining/washing buffers, Fc blocking and background suppression, viability dyes, fluorescent antibodies/chemical probes, fixation/permeabilization reagents, single-color compensation and performance-QC beads.

Core features:

· Stable fluorescence signals: low autofluorescence, low photobleaching, appropriate degree of labeling (D/P), and reproducible linear intensity.

· Low non-specificity and high compatibility: Fc receptor blocking, mild surfactants and protein stabilizers to reduce background; compatible with common laser lines/filter sets.

· Biosafety and viability maintenance: low endotoxin, sterile, maintaining cell viability and morphology; compatible with fixation/permeabilization workflows.

II. Critical Quality Attributes (CQAs)

Quality Attribute	Technical Significance	Test/Validation Method
Fluorescence purity & spectral matching	Ensure clear channel separation and decouplable compensation	Spectral scanning; channel MFI and spillover-matrix evaluation
Degree of labeling (D/P) & activity	Balance signal intensity and antigen affinity	D/P by UV-Vis; antigen-binding curve comparison
Signal-to-noise ratio (S/N)	Reflect sensitivity and background level	MFI ratio of positive/negative populations; isotype/antigen-removal controls
Batch consistency	Cross-batch reproducibility for gating and quantitation	Old vs. new batch in parallel; ΔMFI, CV, and compensation-matrix differences
Non-specific binding	Reduce false positives and threshold drift	Before/after Fc Block; isotype-control background rate
Photostability	Maintain signals during acquisition	Continuous-exposure decay test; time-series MFI
Cell compatibility/viability	Avoid cell damage during staining	PI/7-AAD/fixable viability dyes
Buffer stability	Constant pH/ions/protein to reduce adsorption	pH, conductivity, protein quantitation; long-term in-use observation
Sterility & endotoxin	Prevent cell stimulation and false positives	Plate culture; LAL (as needed)
Fix/perm compatibility	Maintain epitope recognition and fluorescence stability	Signal recovery under different fix/perm conditions

III. Scope of Application

1.Immune cell typing and phenotyping

Identify surface markers (CD molecules) of lymphocytes, monocytes, dendritic cells, etc.

2.Functional assays

Cell proliferation (CFSE, Ki-67), reactive oxygen species (DCFH-DA), apoptosis (Annexin V/PI), cell cycle, etc.

3.Signaling and intracellular antigens

Phospho-proteins and transcription factors (pSTAT, NF-κB) after permeabilization.

4.Drug screening and immune monitoring

Assess drug effects, monitor immunotherapies (CAR-T, immune checkpoints), vaccine responses.

5.Quality control and production monitoring

Purity and viability testing of prepared/cultured cells and flow QC samples.

IV. Main Components and Functional Positioning

Category	Common Reagents/Formulations	Function Description
Buffer systems	PBS, HBSS, FACS buffer (PBS + 1–2% BSA; 0.05–0.1% NaN₃ for fixed/analysis samples only; do not use for live cells/sorting)	Maintain osmolality; reduce non-specific binding (use azide-free for live cells)
Nuclear dyes	DAPI, PI, 7-AAD, Hoechst	Viability and cell-cycle analysis
Fixation/permeabilization	Paraformaldehyde, saponin, Triton X-100	Fix morphology or permeabilize membranes for intracellular staining
Wash & diluent	FACS buffer / PBS	Remove free antibody and background fluorescence

V. Common Problems and Solutions

Problem	Possible Cause	Strategy
Weak signal	Photobleaching, low labeling density, low antigen expression	Use brighter dyes; increase concentration or extend incubation
High background	Non-specific adsorption; excessive dead cells	Add Fc Block; remove dead cells; optimize washing
Spectral spillover	Overlapping dyes; inaccurate compensation	Reset with compensation beads; choose better-separated dye sets
Fluorescence bleaching	Strong light exposure; no anti-fade	Protect from light; shorten run time; add anti-fade
Cell clumping	High Ca²⁺/Mg²⁺ in buffer or unstable temperature	Use Ca²⁺/Mg²⁺-free PBS; keep 4–8 °C
Batch differences	Antibody/dye lot change	Parallel old/new lots; update compensation and gating

VI. FAQs

Q1: Signal too weak after staining; positives hard to resolve?

A: Check antibody dilution and incubation time; confirm target antigen abundance. Try extending incubation (30 → 45 min) at 4 °C in the dark, and ensure wash buffer contains 1% BSA to prevent antibody loss by adsorption.

Q2: Fluorescence drops markedly after fixation?

A: Some dyes are sensitive to aldehyde fixation (especially tandem dyes such as PE-Cy5/PE-Cy7); FITC/APC are generally tolerant to mild fixation. Prefer fixation-compatible or fixable dyes.

Q3: High dead-cell rate and aggregates?

A: Ensure cell health (>90%) before staining; use Ca²⁺/Mg²⁺-free PBS to reduce adhesion; add DNase I (50 µg/mL) to reduce clumping; filter the sample before acquisition to keep particles dispersed.

VII. Usage Specifications and Storage Recommendations

· Storage: 2–8 °C, protected from light and sealed; freeze–thaw can inactivate dyes or aggregate proteins.

· Preparation: Gently mix antibodies; avoid vigorous shaking; equilibrate sample and buffers to the same temperature.

· Light and timing: Acquire promptly after preparing fluorescent antibodies; delay not to exceed 2 h.

· Washing/dilution: Use FACS buffer (PBS + 1% BSA; 0.05–0.1% NaN₃ only for fixed/analysis samples; do not use for live cells/sorting) to maintain low background.

· Instrument standardization: Daily check laser power, spectral response, and compensation with calibration beads.

VIII. Aladdin Product Advantages

1.High-purity fluorescent labeling and rigorous spectral validation

Fluorescent antibodies are verified for single-peak emission with provided excitation/emission parameters, ensuring accurate compensation.

2.Low-background formulation optimization

Contains mild surfactants and Fc-blocking components, markedly reducing non-specific signals from monocytes/macrophages; suitable for complex samples.

3.Batch stability and cross-platform compatibility

Each lot undergoes ΔMFI, compensation-matrix, and multi-platform comparisons (BD, Beckman, Sony), supporting long-term panel use.

4.High biosafety and sterility control

0.22 µm filtration and low-endotoxin processing; compatible with primary cells, immune-cell prep, and preclinical systems.

5.Enhanced photostability and longer preservation

Anti-fade and protein-protection systems maintain signal strength across multiple acquisitions, reducing long-run drift.

6.Complete validation and documentation

Each product provides a COA, spectra, inter-lot comparisons, and recommended compensation matrices, suitable for research and drug-development documentation.

IX. Comparison with Similar Grades

Dimension	Flow Cytometry Grade	IHC Grade	ELISA/CLIA Grade
Target & readout	Suspended single cells; multiparameter fluorescence (MFI), population proportions	Tissue-section localization; chromogenic/fluorescent images and scoring	Solution/solid-phase signals; absorbance/chemiluminescence
Key formulation focus	Fluorescence purity, compensation compatibility, low non-specificity, viability	Antigen retrieval, blocking & chromogenic kinetics, low background	Low baseline, high S/N, linearity and plateau stability
Core reagents	Fluorescent antibodies, FACS buffer, Fc Block, control beads	Retrieval buffers, blockers, polymer secondaries, substrates	Labeled Ab/antigen, substrate systems (TMB/ECL)
Typical QC	Compensation beads, intensity standard beads, isotype controls	Positive/negative paired sections, chromogenic curves, morphology	Negative/positive/blank controls, standard curve (R²)
Common risks	Spectral spillover, photobleaching, non-specificity, lot drift	Over/under-retrieval, tissue detachment, edge darkening	Autofluorescence/chemiluminescence, matrix effects, lot drift
Transfer challenges	Panel design and compensation reuse	Retrieval window and chromogenic timing reproducibility	Curve linearity and clamp-error control
Result form	Single-cell distributions and gating plots	Image localization and intensity scoring	Curve/end-point readouts (OD, RLU)

Reagents suitable for flow cytometry emphasize not only high chemical purity but also spectral accuracy, signal stability, and experimental reproducibility. Through strict control of fluorescent labeling, cross-platform performance validation, and low-background formulation optimization, Aladdin enables each cell analysis to achieve high resolution and data consistency.

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