Equipment
NovoCyte 3000 Flow Cytometer
The NovoCyte 3000 is a high-throughput flow cytometric analyzer with three lasers (Violet 405 nm, Blue 488 nm, Red 642 nm) and filters for detecting up to 13 fluorescent colors. The instrument is equipped with the NovoSampler Pro multi-sampler, which accommodates 24-, 48-, or 96-well plates or a 24-tube rack. The Novocyte uses the NovoExpress software for data acquisition and analysis. Data files can be exported as FCS 2.0 or FCS 3.1.
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 | Default Alias | 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 |
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.