Home › Immunology › Cell Preparation for Flow Cytometry

Steps

1 Determine cell type and sample source 00:05
2 Handle cells at appropriate temperature 01:09
3 Lyse erythrocytes using appropriate buffer 01:27
4 Process samples promptly or preserve them 02:02
5 Remove dead cells and debris 02:57
6 Prepare adherent cells with gentle lifting 03:41
7 Stimulate cells under optimized conditions 04:00

Immunology BD Biosciences

Cell Preparation for Flow Cytometry

Protocol

Difficulty

intermediate

Steps

1

Determine cell type and sample source

Identify what type of cells you are analyzing, such as whole blood, PBMCs, tissue, or cell lines. Consider that rare lymphoid populations benefit from PBMC isolation, while granulocyte analysis requires whole blood.

▶ 00:05

2

Handle cells at appropriate temperature

Keep whole blood at room temperature, but maintain PBMCs and cells in BD effects lacing solution on ice to preserve cell viability and morphology.

▶ 01:09

3

Lyse erythrocytes using appropriate buffer

Use either BD effects lacing solution (which contains fixative) or BD FarmLite buffer (without fixative) to remove red blood cells. Stain for fixative-sensitive antigens before using BD effects lacing solution.

▶ 01:27

4

Process samples promptly or preserve them

Analyze samples immediately after collection to prevent changes in surface phenotype. If immediate processing is not possible, freeze or fix cells to preserve them for later use.

▶ 02:02

5

Remove dead cells and debris

Filter samples through a cell strainer to remove clumping cells and dead cells. Use a viability marker to gate out dead cells and improve analysis quality and population resolution.

▶ 02:57

6

Prepare adherent cells with gentle lifting

For cultured adherent cells, use gentle lifting media such as EDTA or accutase instead of trypsin to prevent cleavage and loss of sensitive surface markers.

▶ 03:41

7

Stimulate cells under optimized conditions

Stimulate cells directly in whole blood or culture PBMCs and tissue-derived cells with appropriate medium, duration, and temperature. Note that phosphorylation assays require only minutes while cytokine assays require hours of incubation.

▶ 04:00

🚨 Failure Case Library (21) + Submit your own case

Excessive Autofluorescence in Solid Tissue Samples

Solid tissue samples display much higher autofluorescence than PBMCs across multiple channels. Tissue-derived cells show elevated background due to structural proteins, extracellular matrix components, and pigments.

💡 5 · ✓ 5

Incomplete cellular fixation due to insufficient incubation

Cells show incomplete crosslinking when fixation time is inadequate, resulting in poor preservation, continued enzymatic activity, or inadequate biosafety inactivation of infectious samples. Fixed cells demonstrate degradation during storage or analysis.

💡 4 · ✓ 5

Inadequate Pathogen Inactivation in Infectious Samples

Samples from infected or potentially hazardous sources show signs of incomplete inactivation, creating biosafety concerns during handling and flow cytometry analysis. Validation assays indicate residual infectious potential.

💡 4 · ✓ 6

High Background Signal from Autofluorescent Cell Types

Elevated background fluorescence intensity across multiple channels, particularly affecting green (FITC) and orange (PE) channels. Difficult to distinguish specific antibody staining from background noise.

💡 5 · ✓ 5

Aldehyde-Based Fixation Amplifying Autofluorescence

Fixed cell samples exhibit dramatically elevated autofluorescence compared to live cells, particularly in green and UV channels. Background signal increases after fixation protocol, reducing signal-to-noise ratio.

💡 4 · ✓ 5

Isotype Control Signal Is Abnormally High

The isotype control antibody shows unexpectedly high fluorescence signal, making it difficult to distinguish true positive staining from background in flow cytometry analysis.

💡 6 · ✓ 6

Excessive Background in Myeloid-Rich Cell Populations

Samples enriched for monocytes, macrophages, or dendritic cells show uniformly high fluorescence across all antibody channels including isotype controls, obscuring specific marker detection.

💡 4 · ✓ 5

Loss of Antibody Signal After Pre-Fixation

Antibodies show complete or partial loss of fluorescent signal when cells are fixed with 4% PFA before antibody staining. Target cells that should be positive appear negative or dim compared to unfixed controls.

💡 5 · ✓ 6

Incorrect Positive/Negative Cell Population Ratios

The measured ratio of positive to negative cells for a given marker appears inaccurate or inconsistent. Background signals are not correctly measured, leading to improper gating and incorrect population quantification.

💡 4 · ✓ 4

Poor Resolution of Dim Markers Masked by Autofluorescence

Low-expression markers become indistinguishable from background. Positive and negative populations show poor separation, with dim fluorophores completely masked by cellular autofluorescence.

💡 4 · ✓ 5

Fluorescence Spillover into Secondary Detectors

One fluorochrome's emission spectra spills over into another detector channel, creating false positive signals. Data appears contaminated with signals that do not represent true marker expression.

💡 4 · ✓ 4

High Non-Specific Binding After Cell Fixation

Following fixation with formaldehyde or paraformaldehyde, both test antibodies and isotype controls show elevated background signal and increased non-specific staining patterns.

💡 4 · ✓ 5

Elevated Background Fluorescence Post-Fixation

Flow cytometry analysis reveals increased non-specific fluorescence across all populations following fixation. Negative control cells show elevated signal, reducing signal-to-noise ratio and obscuring true positive events.

💡 4 · ✓ 4

Cell Morphology Distortion and Loss of Scatter Properties

Forward scatter (FSC) and side scatter (SSC) profiles show abnormal patterns after fixation. Cell populations cluster abnormally, size measurements are inconsistent, and gating strategies based on morphology fail to resolve expected populations.

💡 4 · ✓ 4

Workflow disruption from inappropriate fixation timing decisions

Experimental workflow becomes incompatible with chosen fixation strategy, leading to scheduling conflicts, sample degradation, or compromised data quality. Researchers cannot complete multi-step protocols in required timeframes.

💡 4 · ✓ 5

Progressive Biological Changes in Unfixed Time-Course Samples

Time-course experiments show artifactual progression or decay of signals when samples are not fixed at each timepoint. Biological processes continue during sample handling, obscuring true temporal snapshots.

💡 5 · ✓ 6

Non-specific Antibody Binding Creating Noise

Antibody binds non-specifically to cells of interest, resulting in noisy data and elevated background. True marker expression cannot be distinguished from non-specific binding events.

💡 4 · ✓ 5

Suboptimal PFA concentration causing inadequate or excessive fixation

Using incorrect paraformaldehyde concentration results in either incomplete cellular preservation (too low) or excessive epitope masking and fluorophore damage (too high). Standard flow cytometry protocols show inconsistent results across experiments.

💡 4 · ✓ 6

Inconsistent Fixation Quality from Incorrect PFA Concentration

Samples show variable fixation quality, with some cells over-fixed (high autofluorescence, poor staining) and others under-fixed (continued biological activity). Reproducibility across experiments is compromised.

💡 5 · ✓ 6

Background Spread Due to Spillover Not Corrected

Even after compensation, background spread from spillover effects makes it difficult to determine appropriate gate boundaries. Positive and negative populations are not clearly separated in the detector of interest.

💡 4 · ✓ 4

High Background in Biological Control Sample

Biological control (e.g., unstimulated sample in stimulation assay) shows unexpectedly high background, making it difficult to set clear positive/negative boundaries.

💡 4 · ✓ 5

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