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Omid Khalafi: Gating Strategy and How Do We Decide Which Cells to Analyze
Omid Khalafi/LinkedIn
Sep 21, 2026, 15:49

Omid Khalafi: Gating Strategy and How Do We Decide Which Cells to Analyze

Omid Khalafi, Technical Support Specialist and After Sale Service Manager at Pharmateb, shared a post on LinkedIn:

“Gating Strategy

How Do We Decide Which Cells to Analyze?

A flow cytometer can collect tens of thousands of events.

But not every event is relevant.

Some may represent:

  • Debris
  • Cell fragments
  • Dead cells
  • Doublets or aggregates
  • Unwanted cell populations

So before interpreting fluorescence markers, we need to answer a fundamental question:

Which events should actually be analyzed?

This is the purpose of gating.

What is a gate?

A gate is a defined region on a flow-cytometry plot used to select a population of events for further analysis.

Think of it as a filter:

All events, then selected events, then more specific population and final analysis

The important point is that gating is not simply ‘drawing a box around the cells.’

It is a logical sequence of biological decisions.

1. Start with the right population

A common first step is identifying the main cell population using parameters such as: FSC × SSC

This can help distinguish cells from debris and provide an initial view of lymphocytes, monocytes and granulocytes.

But FSC/SSC alone does not automatically identify a cell type.

It only gives us information about its physical characteristics.

2. Exclude unwanted events

Before analyzing markers, we may need to remove: debris, then dead cells and doublets

For example, doublet discrimination commonly uses relationships such as: FSC-A vs FSC-H

The goal is to ensure that an event represents a single cell, rather than two cells passing the laser together.

3. Apply biological markers

Once the appropriate population has been selected, fluorescence markers can progressively narrow the analysis.

For example: All events, Leukocytes, Lymphocytes, T cells, CD4⁺ T cells

Each step answers a more specific question.

The danger of a bad gate.

A gate can create a very convincing result while still being wrong.

If the initial gate excludes part of the population, everything downstream can be affected.

This is especially important because: A downstream result can only be as reliable as the population selected upstream.

Gating is not universal

There is no single gating strategy that works for every sample.

The appropriate strategy depends on:

Sample type plus clinical question plus expected biology plus markers and controls

A strategy appropriate for peripheral blood may not be appropriate for bone marrow, CSF, or a tissue sample.

Good flow-cytometry analysis does not start with:

‘Where is the positive population?’

But starts with: Which cells am I looking at?

Then: What population should I analyze next?”

Omid Khalafi: Gating Strategy and How Do We Decide Which Cells to Analyze

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