FlowJo Data Analysis Software Operation

severe

Rare Cell Population Incorrectly Gated or Missed

Target rare immune cell subsets (e.g., dendritic cells, innate lymphoid cells, hematopoietic progenitors) appear overestimated or masked by abundant terminally differentiated cells. Gating on single markers yields incorrect population percentages (e.g., 4.5% vs. true 1.2% DCs).

💡 4 · ✓ 4

severe

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

severe

Incorrect Use of Isotype Controls for Gating Dim Markers

Gates set using isotype controls for dim or activation markers result in inaccurate population identification, with either false negatives (missed positive cells) or false positives.

💡 4 · ✓ 5

moderate

Autofluorescence Complicating Cell Population Gating Strategy

Difficult to establish clear gates between positive and negative populations. Autofluorescent cells appear in unexpected regions of scatter plots, creating ambiguous boundaries and potential misidentification of cell populations.

💡 4 · ✓ 5

moderate

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