No/Low Fluorescent Signal

No fluorescence in target cell population or signal close to negative control

1. Insufficient cell permeabilization (for intracellular antigen detection);

2. Fluorochrome quenching or low labeling efficiency;

3. Low cell concentration or antigen expression;

4. Antibody inactivation, mismatched species or over-dilution;

1. Permeabilize cells with 0.1% Triton X-100 for 10–15min for intracellular staining, omit for membrane protein detection;

2. Operate in dark throughout and use high-activity fluorochromes;

3. Concentrate cells to 1×10⁶–1×10⁷ cells/mL or use signal amplification reagents;

4. Verify antibody validity, optimize dilution ratio (1:50–1:200) and match target cell species;

High Non-specific Background

Elevated fluorescence in negative cell population, unclear population separation

1. Excessive antibody concentration;

2. Inadequate blocking (Fc receptor interference);

3. Excessive cell debris/dead cells causing non-specific antibody adsorption;

4. Incomplete washing with residual unbound antibody

1. Reduce antibody concentration

2. Block with 1% BSA or species-matched serum for 15–20min, add Fc receptor blocker for Fc-positive cells;

3. Filter cells through nylon mesh before assay, exclude dead cells with trypan blue/7-AAD;

4. Wash with PBS containing 2% FBS, discard supernatant completely after centrifugation and repeat 3 times

Cell Aggregation

Massive clump signals in scatter plot, unable to gate accurately

1. Over/under-digestion of adherent cells;

2. Insufficient pipetting for cell dispersion;

3. Excessively high centrifugation speed causing cell compression;

4. Inadequate mixing during antibody incubation

1. Optimize digestion conditions (e.g., trypsinize until cell edge retraction), avoid over-digestion;

2. Pipette cells gently with Pasteur pipette or pass through 200-mesh sieve;

3. Reduce centrifugation speed (300–400×g for 5min);

4. Mix gently by vortex or incubate on shaker at low speed

Low Resolution, Poor Population Separation

Overlap between target and miscellaneous populations, unable to distinguish

1. Improper fluorescence compensation setting;

2. Low laser power reducing detection sensitivity;

3. Overlapping emission spectra of antibody fluorochromes;

4. Poor cell culture status with uneven antigen expression

1. Adjust fluorescence compensation with single-stained tubes to eliminate inter-channel interference;

2. Increase laser power appropriately (avoid excessive cell damage);

3. Select fluorochrome combinations with non-overlapping emission spectra (e.g., FITC/PE/Cy5);

4. Use cells in logarithmic growth phase and standardize culture conditions

Low Cell Viability

Dead cell ratio ＞ 20% during detection

1. Prolonged sample processing time causing hypoxia-induced cell damage;

2. Severe centrifugation/pipetting leading to mechanical damage;

3. Improper osmotic pressure of staining solution causing cell rupture;

4. Toxic effects of antibodies/reagents

1. Shorten sample processing time (<2h) and operate on ice throughout;

2. Pipette gently and keep centrifugation speed below 500×g;

3. Prepare staining solution with isotonic PBS and avoid high-concentration reagents;

4. Select low-toxicity antibodies and optimize incubation time (15–30min)

Poor Experimental Reproducibility

Large variations in results between batches and obvious fluctuation in cell proportion

1. Inconsistent staining temperature/time;

2. Unstable flow rate causing data acquisition errors;

3. Different initial cell status (passage/density variations)

1. Fix staining conditions (incubate at 4℃ for 30min in dark);

2. Calibrate instrument before detection and maintain stable flow rate (100–300 events/sec);

3. Use cells of the same passage and in logarithmic growth phase