Imaging Flow Cytometry and Flow Cytometric Analysis Core

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NovoCyte 3000 Flow Cytometer

The flow cytometer examines visible light scatter and fluorescence parameters for each cell. Forward scatter indicates the relative size of the cell, while side scatter indicates its internal complexity. Flow cytometry can measure the number of cells, the percentage of live cells, and the presence of intra- and extra-cellular markers. A few common applications are cell cycle analysis, apoptosis assays, and phenotyping of mixed cell populations.

When planning a flow cytometry experiment and choosing fluorochromes, it is important to know the fluorochromes' specific excitation and emission wavelengths, as well as the lasers, filters, and detectors available on the instrument. Below is the information for the NovoCyte 3000.

NovoCyte 3000 Configuration and Fluorochromes
Excitation Laser	Channel	Bandpass Filter	Ideal dyes	Examples of Fluorochromes
Violet 405 nm	V445	445/45	Pacific Blue	DAPI, BV421, eFluor 450
	V530	530/30	AmCyan	BV510, cFluorV547, AF430, eFluor506
	V572	572/28	Pacific Orange	QDot565*, BV570
	V615	615/20	Qdot 605	BV605, eFluor 605NC
	V675	675/30	Qdot 655	BV650, eFluor 700
	V780	780/60	Qdot 800	BV785, Qdot 800*
Blue 488 nm	B530	530/30	FITC	FITC, AF488, CFSE, GFP, YFP, BB515
	B572	572/28	PE	PE, Cy3, eFluor 570, SYTOX Orange
	B615	615/20	PE-Texas Red	PI, PE-Texas Red, RFP
	B675	675/30	PerCP	BB660, PE-Cy5,* PI, 7AAD, PerCP-Cy5.5* PE-AF647
	B780	780/60	PE-Cy7	PE-Cy7, PE-AF750
Red 640 nm	R675	675/30	APC	APC, AF647, AF660, eFluor660, DRAQ 5
Red 640 nm	R780	780/60	APC-Cy7	APC-Cy7, APC-AF750, APC-H7, APC-eFluor780

It is important to remember that many fluorescent dyes are excited by more than one laser and fluoresce in multiple channels. It is very helpful to use a spectral viewer when planning an experiment.