How to use this tool — CHOP Flow Cytometry Core

Two pages, two jobs

Instrument configurations is the reference page: it shows every instrument in the lab with its lasers, its detectors, and which fluorochrome each detector is meant for.
Instrument Selector is the planning tool: enter a list of dyes and it tells you which instruments can detect them and in which detector each dye lands. For the spectral instruments (Aurora and Opteon) it also tells you which pairs of dyes will be hard to tell apart.

A short guide to the Instrument Selector

There are two ways to enter a panel. Paste a whole list into the large box — commas, tabs, pipes and new lines all work. Or type a name into the smaller box and pick from the suggestions that appear: click one to tick it and it goes into your panel immediately, click it again to take it back out, and tick as many as you like. Click anywhere outside the list to close it.

The Add button does something different: it enters the text exactly as you typed it. That is how you get a dye the tool does not know into your panel. It is greyed out while the suggestion list is open, because at that moment it would add your search text — a bare BUV or Alexa Fluor — and not the dyes you have ticked. Close the list and it comes back.

Copy panel and Clear all sit together under your list of dyes.

The tool generates a collapsible card for each instrument, and a row of badges on each card shows at a glance whether that instrument can detect your panel, or where the problems are.

Undetectable: 1 Suboptimal: 2 Unresolvable: 1 Not in library: 1 Detected: 12/14

This tool does not design panels. It does not know your markers, your antibodies, or how autofluorescent your cells are — it only tells you which instrument can detect a given set of fluorochromes.

The badges on each card

Badge	What it means
Undetectable: 1	No detector sees this dye. It cannot be used on this instrument.
Suboptimal: 2	Seen, but only a small share of the dye's light reaches a detector, or the detector sits off to one side of the dye's emission. Shown struck through and not counted as detected. Can still work when the positive events are bright and clearly separated from the negative population.
Unresolvable: 1	Two dyes in your list that this instrument cannot tell apart.
Not in library: 1	Dyes the tool does not recognise — see below.
Detected: 14/14 ✓ Detected: 12/14	How many of your dyes this instrument sees clearly. Green with a tick when it sees all of them.

Hover any badge for the list of dyes it is counting.

“Please note” — a dye on the wrong laser

Some cards carry a Please note paragraph below the badges. It appears when a dye is detected on a laser it was not designed for — for example BV570, a violet dye, read off the 561 nm laser on an instrument with no violet line. The dye is still detected and still counted; it will simply be dimmer than you are used to, and the note tells you which laser gives the strong signal.

The note asks for a laser and a matching detector, because both are needed and the two do not always come together: an instrument can carry the right laser and still have no detector that reaches the dye's emission, which is why the dye ended up on the wrong laser here in the first place.

This is information, not a warning against using the dye. Whether the weaker signal matters depends on your sample, which the tool cannot know.

How hard is this pair to separate?

On the spectral instruments (Aurora, Aurora CS, NovoCyte Opteon) you can open a similarity matrix under the channel grid. Each cell scores one pair of your dyes: 0 means nothing alike, 1 means identical. Every cell is on the same scale, so 0.94 means the same thing everywhere in the table. Hover a cell for the names and the advice.

Value	What to do
< 0.80 Easy to resolve, recommended	No reason to avoid this combination. Lower is better still — if you have a choice between a pair at 0.10 and a pair at 0.50, take the 0.10.
0.80 – 0.90 Resolvable, avoid on co-expressed markers	Fine in most panels. Think twice if both markers appear on the same cells, or if either is dim.
0.90 – 0.98 Difficult to resolve, avoid if possible	Workable only if the pair really matters and both markers are bright and well separated biologically. Test it before you commit.
≥ 0.98 Unresolvable, do not use together	Replace one of the two. Unmixing will not separate them.
n/a	One of the two is not in the library, so the pair cannot be compared.

Every SI number is an estimate. Each experiment is different — different reagents, different cellular autofluorescence. Your own data can be right even when it disagrees with the values here, so test any pair you are going to depend on.

The ImageStream works differently

On every other instrument the lasers and detectors are fixed, and a dye lands where the optics put it. The ImageStream has twelve channels shared between fluorescence, brightfield and side scatter, and you decide which lasers are on and which channels record the brightfield and side scatter signals. The same panel can therefore land in different channels depending on how the instrument is set up.

The ImageStream card carries a configuration strip matching what you will set in Inspire. Change anything on it and your panel is re-placed straight away.

Control	What it does
Lasers 405 · 488 · 561 · 642	Turn a line off and the dyes that need it move to a weaker channel or drop out. Channels 1–6 are fed by 488 and 561; channels 7–12 by 405 and 642.
Brightfield	Takes one channel on each camera. Those two are then unavailable for dyes.
Side scatter Ch6 or Ch12	Costs one channel and nothing else — the 785 nm line never excites a dye.

All three do the same thing: they take channels away from your panel. A dye whose best channel is taken moves to its next-best on its own; if nothing suitable is left it is reported as undetectable with the reason given. Moves are listed under Relocated, so a dye that shifted looks different from one that was always there.

The Autoconfigure button turns on the lasers your panel needs and sets the brightfield channels so that as many fluorochromes as possible are detected. Side scatter is optional and is turned on only when it does not take a channel a fluorochrome needs. Autoconfigure keeps choosing for you until you move a control yourself; after that the setup is yours and adding another dye will not change it back.

More lasers is not better. An unnecessary laser line adds background and increases spillover. Autoconfigure turns on only the lines your panel actually requires — and switching a line on or off can change which detector a fluorochrome is listed in.

A dye followed by a small * is one the manufacturer lists as working on the ImageStream even though the excitation or the channel filter is far from ideal. BV421 is the clearest case: it is detected well in Channel 7 even though that channel's 435–505 nm filter misses its emission peak, because BV421 is very bright. Dyes the manufacturer lists as not usefully detected are left out of the ImageStream channel listings altogether.

There is no similarity matrix for the ImageStream or for the conventional cytometers. Similarity indices are calculated only for the spectral instruments (Aurora and Opteon).

Dyes the tool does not know

Anything not in the library — pasted in, or typed and entered with Add — shows as a highlighted chip and is counted under Not in library. It is not placed and not included in any other count.

A panel can look clean while one of its dyes was never checked at all. Read the Not in library badge before you trust Detected.

On conventional cytometers you can place it yourself: click the chip, then click the channel you expect it in. It is then checked for conflicts like any other dye.
On spectral instruments that does not help — without a spectrum the pair cannot be compared, so its matrix cells read n/a.

If a dye you use regularly is missing, email flowcytometry@chop.edu and it can be added.

Worth knowing

Placement is where a dye peaks, not the only place it is seen. On a spectral instrument two dyes peaking in the same channel is common and usually harmless — the similarity matrix, not the channel, tells you whether a pair is a problem.
The Aurora and the Aurora CS are optically identical. The difference is that one sorts.
The card order never changes as you edit your panel, so an instrument stays where you last saw it.
Everything runs in your browser. Nothing is sent anywhere, and it works offline.

When you need more than this

The tool answers "will this run, and where". It does not titrate, does not know your antigens or how strongly they are expressed, and cannot predict how your particular tandem will behave. For anything beyond a dye list, talk to the core: flowcytometry@chop.edu · 215-590-3402.

Reporting a problem? Please include the version line at the foot of the page you were looking at.