BenchMasteryMolecular biology techniques & virtual lab

Flow cytometry & FACS

Surface markers are stained on live cells; intracellular targets (cytokines, transcription factors, phospho-proteins) need fixation and permeabilisation first, which changes scatter and some epitopes.

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Surface & intracellular staining

Surface markers are stained on live cells; intracellular targets (cytokines, transcription factors, phospho-proteins) need fixation and permeabilisation first, which changes scatter and some epitopes.

How it differs from the standard method

  • Stain surface markers and viability dye before fixing.
  • Use the permeabilisation buffer suited to the target (e.g. methanol for phospho-epitopes).
In this lesson · about 1 min read
  1. Measuring cells one at a time
  2. Spillover and compensation
  3. Sorting
  4. Key terms
  5. When to use it
  6. Sub-techniques

1Measuring cells one at a time

A flow cytometer pushes cells in single file through laser beams. For every cell it records forward scatter (roughly size), side scatter (internal complexity or granularity) and the fluorescence of each antibody-bound dye in separate detectors.

FSC / SSC

Scatter

Separates lymphocytes, monocytes, granulocytes and debris without any stain.

fluorescence

Markers

Antibodies coupled to fluorochromes report which proteins each cell carries and how much.

% of parent

Gating

Successive gates (cells → single cells → live → lineage → subsets) give population frequencies.

2Spillover and compensation

Fluorochromes have broad emission spectra, so some FITC light lands in the PE detector. Uncorrected, bright FITC⁺ cells appear PE⁺ too, producing a diagonal smear.

3Sorting

A sorter vibrates the stream so it breaks into droplets; when a droplet holds a cell inside the sort gate, it is charged and deflected into a collection tube. Purity, recovery and viability trade off against speed: faster rates mean more droplets carrying two cells.

Key terms

Practise these as flashcards →
Gate

A region on a plot that selects a population for further analysis.

Compensation

Mathematical removal of each dye's spillover into other detectors, based on single-stained controls.

FMO control

All stains minus one; defines the boundary between negative and positive for that marker.

Titration

Testing antibody amounts to find the one with the best separation between positive and negative cells.

Viability dye

A dye that enters dead cells (with leaky membranes) so they can be gated out.

MFI

Median fluorescence intensity of a population; a measure of how much antigen cells carry.

When to use it

  • Counting immune-cell subsets in blood or tissue, or checking marker expression after transfection or treatment.
  • Measuring cell-cycle stage, apoptosis or proliferation cell by cell.
  • Isolating live, defined populations for culture or sequencing (FACS).

Compared with alternatives

Immunofluorescence →

Shows where a protein is inside a cell; flow gives numbers for thousands of cells but no images.

Cell counting & viability →

Simple counts and viability without markers.

Apoptosis assays →

Annexin V/PI staining is a flow assay.

Single-cell RNA-seq →

Profiles the whole transcriptome per cell instead of a handful of proteins.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Surface markers are stained on live cells; intracellular targets (cytokines, transcription factors, phospho-proteins) need fixation and permeabilisation first, which changes scatter and some epitopes.

  • Stain surface markers and viability dye before fixing.
  • Use the permeabilisation buffer suited to the target (e.g. methanol for phospho-epitopes).