Antibodies for flow cytometry: building a panel that works

Building the panel

1. Brightest fluorochrome on the dimmest antigen. Resolution depends on the separation between positive and negative, so a dim antigen needs a bright fluorochrome to be seen at all, and a highly expressed antigen can afford a dim one. Getting this backwards is the single most common panel design error.
2. Check the instrument configuration first. A fluorochrome is only usable if the instrument has a laser to excite it and a detector to read it. Get the configuration and build the panel against it, because a beautiful panel on paper for an instrument that cannot read half of it is wasted money.
3. Spillover and where it lands. Emission spectra overlap, and the spillover from a bright fluorochrome degrades resolution in the channels it reaches. Avoid putting two bright dyes with heavy mutual spillover on two dim, co-expressed antigens, and expect compensation to cost resolution rather than restore it.
4. Clone matters as much as the target. Different clones against one antigen recognise different epitopes, and a fixation or permeabilisation step can destroy the epitope one clone needs. Choose the clone from published use in your application, not simply from the target name.
5. Titrate every antibody. The manufacturer's suggested volume is a starting point on their cells in their buffer. Titrating on your own cells finds the concentration giving the best separation, saves reagent, and reduces background. This is the step that most improves a panel and the one most often skipped.

Controls belong in the reagent budget

Single stain controls for compensation, a viability dye, and fluorescence minus one controls wherever a gate is not obvious are part of the panel rather than optional additions. They consume antibody and instrument time, so they belong in the budget from the start.

Dead cells bind antibody non-specifically and generate false positives across every channel, which is why a viability discriminator is not optional in any panel that matters.

FACS antibodies: lots, storage and reproducibility

Conjugated antibodies vary between lots and degrade with light exposure and freeze and thaw cycles. Store them as the manufacturer says, keep them dark, and never freeze a conjugate unless the supplier says to.

For a study running months, buy enough of one lot to finish or plan a bridging comparison against a retained aliquot. Lot changes mid-study move the numbers in ways that look biological.

A cyclin e antibody and a narrow window

Cyclin E peaks at the transition into S phase and is degraded quickly, so a cyclin e antibody reports a cycle position and an asynchronous population averages it away. Synchronisation and a stated harvest time are the method. Because it is degraded by a ubiquitin pathway, a proteasome inhibitor lane is what shows the antibody works when the protein is scarce.

A plk4 antibody and a very low abundance kinase

PLK4 controls centriole duplication and is present at very low levels, so a plk4 antibody usually needs an overexpression or an inhibitor treated positive control to show it works at all. It is autoregulated by degradation, so a stabilising treatment raises it. The readout of interest is centriole number by imaging rather than a band on a blot.

A cdk9 antibody and a transcriptional kinase

CDK9 phosphorylates the polymerase tail rather than driving the cell cycle, so a cdk9 antibody belongs in transcription work and its readout is the polymerase's serine 2 phosphorylation. It sits in a complex with a cyclin partner, which an immunoprecipitation brings down. Its own level is stable, so an inhibitor treated lane is what demonstrates the pathway.

A polq antibody and a specialised polymerase

POLQ, the theta polymerase, carries out microhomology mediated end joining and is a synthetic lethal target, so a polq antibody is used to confirm loss or knockdown rather than to measure a response. It is a large protein, which makes transfer the usual failure, and a patient or engineered line with known loss is the cleanest negative control.

A parp2 antibody and the paralogue question

PARP2 shares catalytic sequence with PARP1 and is far less abundant, so a parp2 antibody needs cross reactivity data against it and a knockout lysate to make a band attributable. Since most inhibitors hit both, a functional claim about one requires genetics rather than pharmacology, and that is what a figure using this reagent has to show.

Common questions

How do I match fluorochromes to antigens?

Put the brightest fluorochromes on the dimmest antigens, because resolution depends on separating positive from negative. Check every choice against the instrument's lasers and detectors before ordering.

Do I need to titrate every FACS antibody?

Yes. The manufacturer's suggested volume was determined on their cells in their buffer. Titrating on yours improves separation, reduces background and saves reagent.

Why does the clone of a flow cytometry antibody matter?

Different clones recognise different epitopes on the same antigen, and fixation or permeabilisation can destroy the epitope a given clone needs. Choose the clone from published use in your application.

Which controls are essential?

Single stain controls for compensation, a viability dye to exclude dead cells that bind non-specifically, and fluorescence minus one controls wherever a gate boundary is not obvious.

Browse by service class

• Compare Gene synthesis on advertised prices