The NCSU Flow Cytometry and Cell Sorting Laboratory is located on the third floor of the main CVM building. The laboratory provides instrumentation and assistance for multi-parameter flow cytometric analysis and sorting. It is available for use to all NCSU researchers and to those outside the NCSU community and operates as a by appointment, fee for service laboratory.

Instrumentation and Policies

Analyzers

Becton Dickinson LSRII

Has three lasers, 488 nm, 635 nm and 405 nm. Can analyze up to nine colors and utilizes BD Diva software for acquisition. Scheduling for the LSRII is available by online invitation calendar. Click here to access the LSRII configuration for experiment planning.

Beckman Coulter CytoFlex

The Beckman Coulter CytoFlex has a 488nm laser as well as a 633nm laser. It has the ability to analyze 3 parameters off the 488nm laser and one parameter off the 633nm laser. The CytoFlex is customizable for specific experiments. Scheduling is available by online calendar.

Cytek modified BD FACScan

Has been modified with a 635 nm laser and two additional PMTS to provide a total of five colors from the 488 nm and 635 nm lasers. Utilizes FlowJo Collection Software for acquisition and FlowJo for analysis.

High Speed Cell Sorter

Dako Cytomation MoFlo

The Cytomation Inc. MoFlo (Modular Flow cytometer) provides our sorting capability. In addition, the MoFlo provides the capabilities for the more complicated experiments that can not be done on the FACScan or FACSCalibur (e.g. more than four colors simultaneously), other fluorochrome emission wavelengths, and requirements for other laser excitations (e.g. ultraviolet). Our MoFlo is configured with 1 iCyte 200mw Blue Sappire 488nm laser and a Melles Groit Helium-Neon laser.

The He-Ne produces 35mW of 633nm light. We also have a UV laser that is tunable but normally operates at 350nm. It is possible to tune this laser to the following wavelengths: 350nm, 407nm, 490-530nm, and 568nm. The MoFlo is capable of analyzing up to 10 colors plus forward and side scatter. In terms of sorting capability, the

MoFlo can sort for up to four different cell populations at once and is capable of purifying populations that are less than 1% of the total input population. The MoFlo was the first commercial instrument developed with high speed sorting. We have done sorts as high as 50,000cells/sec with recovery rates of 90% or greater. Typical sorts on our MoFlo result in purified populations of 95% or greater. We have the ability to sort a wide variety of cell types due to the variable sizes of our interchangeable nozzle tips.

We can sort materials that range in size from DNA to material (e.g. cells, bacteria, yeast) that is up to 150 um in size. The MoFlo can sort into a variety of capture vessels ranging from 1.5ml microcentrifuge tubes to 15ml to 50ml conical centrifuge tubes. In addition to test tubes typically used for bulk sorts, the Cyclone on the MoFlo can deposit variable number of cells into a variety of catch vessels; 6, 12, 24, 48, 96, 384 well cluster plates as well as onto slides. It is also possible to sort cells directly onto filters or nitrocellulose membranes. Individual cells can be sorted into plates or slides for cloning for hybridoma production or PCR analysis or in-situ analysis.

Computers and Software

The LSRII uses a PC computer and FACSDiva software. The FACSCalibur uses a Macintosh G5 computer with BD Cell Quest Pro software. The MoFlo is operated using a PC computer with Summit software. In addition, the laboratory has 1 Macintosh auxiliary analysis station.

In addition to Cell Quest Pro and Diva software, FlowJo and FCS Express software is also available.

FCS Express can be downloaded on your personal computer to do analysis in your lab. For instructions, please contact Sarah Bianco.

Requirements

Necessary information before bringing samples to the flow cytometry and cell sorting facility:

Appointments

Users must schedule appointments directly with the operator (Sarah Bianco) as far in advance as possible for sorting and facility assisted experiments.

Users who have been trained will be given access to an online calendar system where they can schedule appointments.

All appointments require 24 hour notice for cancellations. If you do not notify Sarah Bianco in person or in writing, you will be charged.

For first-time facility users, it is advisable to make an appointment with the operator to discuss your proposed experiment to be sure the laboratory has the capability of acquiring the information you wish.
Please contact Sarah Bianco to make an appointment.

Fees

Instrument use fees are charged by the hour in 15-minute increments for laser time. Charges will begin at the time of the appointment.

Analyzers

MoFlo (only the facility operator is allowed to run the MoFlo)

Other Fees

Flow Cytometry Core Manager

Ms. Sarah Schuett, Manager
Phone: 919.513.6443
Email: seschuet@ncsu.edu