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
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.
Necessary information before bringing samples to the flow cytometry and cell sorting facility:
- Charges for experiments will begin at the time of the appointment. Clients will be charged for the time if they are late unless they provide 4 hours telephone notice as to the time change. It is important to think about how long your sample will take to prepare and plan accordingly. The facility is extremely busy and delays mean others can’t use the equipment.
- Clients must give 24 hours notice of cancellation or they will be charged for the experiment.
- For sorting experiments, client should bring tubes and medium to sort into. It is best not to sort into an empty tube. Antibiotics are recommended for media to be sorted into. Facility personnel can provide information as to the appropriate tubes to bring. Samples for sorting should be at 20-30 million cells per milliliter of media.
- Please make sure to bring all appropriate negative and single color controls for your experiments. Cells only tubes are useful for eliminating background floursescence, especially with cell-cultured cells. If you have questions about your controls, please contact Sarah Bianco for assistance.
- Clients with transfected samples should always bring an untransfected sample for comparision.
- All experiments for the analyzers must be brought in BD Falcon polystyrene round bottom tubes-12 x 75 mm style. These can be purchased from Fisher Scientific under state contract. The BD number is 352054. If polystyrene tubes are a problem for your cells of interest, please contact facility personnel for assistance.
- For sorting, it is possible to sort from a number of tubes-microcentrifuge tubes, 5ml round bottom tubes, 15 or 50 ml conical centrifuge tubes. Cells can be sorted into the same variety of tubes.
- Procedure for flow cytometry prep for blood
- Dilute antibodies and prepare sample tubes with appropriate amount of antibodies in each tube. This is usually 100 uls of diluted antibody per antibody per tube. It is possible to mix directly conjugated antibodies in the tube together for staining.
- Make sure to prepare at LEAST ONE CELLS ONLY TUBE and one or more negative control tubes based on how many different isotypes of antibodies used and single color staining tubes if this is the first time these antibodies have been run by this flow operator. Consult with the individual flow operator before setting up experiment to see what they prefer.
- Collect blood in appropriate anticoagulant. You will need 100 uls of blood suspension per tube for each specimen. Make sure to run CBCs if total cell numbers are needed for interpretation of flow data. Spin the blood at sufficient speed to pellet cells(usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins). Remove plasma and save. Add PBS of Hanks Balanced Salt Solution in sufficient quantity to bring the blood back to its original volume.
- Add 100 uls of blood suspension to each sample tube.
- Place tubes on a rotating shaker on moderately fast shake, at room temp, in the dark for 20-30 mins. If there are questions about your cell surface markers, incubate antibodies and blood on ice on shaker. If incubating for longer periods, place on ice or at 40 C in dark. Blood & antibody tubes make be refrigerated overnight. Cover and keep in the dark. Overnight incubations should also have 2% fetal calf serum added to them.
- Wash each tube with 3-4 ml PBS and spin to pellet cells (usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins). Carefully pour off supernatant PBS(until red cells come just to the rim of the tube) or enough not to loose any pelleted cells.
- Resuspend cells by racking on plastic rack in resuidual liquid.
- FOR DIRECTLY CONJUGATED ANTIBODIES:
For Blood: Lyse red blood cells using BD FACS 10X lysing solution (IF YOU HAVE A BD MACHINE-USE COULTER SOLUTION FOR COULTER MACHINES) diluted to 1X with ddH2O. Lysing needs to be done twice using one ml of diluted lysing solution for each and washing with 2 ml of PBS centrifuge to pellet cells (usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins)between each lysing To Lyse, place tubes on a rotating shaker on fast shake, at room temp, for 10 mins for each lysing. After second lysing wash tubes with 2 ml of PBS and centrifuge to pellet cells (usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins) Resuspend cells in 200 ul of PBS w/2% fetal calf serum or media and run on Flow Cytometer.
- FOR UNCONJUGATED ANTIBODIES:
After incubation, wash tubes as before. For tissue samples: resuspend in 200 uls of PBS w/2% CSS or media and run on Flow Cytometer. For Blood: Lyse red blood cells using BD FACS 10X lysing solution (IF YOU HAVE A BD MACHINE-USE COULTER SOLUTION FOR COULTER MACHINES) diluted to 1X with ddH2O. Lysing needs to be done twice using one ml of diluted lysing solution for each and washing with 2 ml of PBS centrifuge to pellet cells (usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins) between each lysing. To Lyse, place tubes on a rotating shaker on fast shake, at room temp, for 10 mins for each lysing. After second lysing wash tubes with 2 ml of PBS and centrifuge to pellet cells (usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins). Resuspend in 200 uls of PBS w/2% CSS or media and run on Flow Cytometer.
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.
Instrument use fees are charged by the hour in 15-minute increments for laser time. Charges will begin at the time of the appointment.
- Operator-conducted acquisition and analysis – $150/hr
- User-conducted acquisition and analysis (supervised) – $50/hr
- 30 minute minimum charge. After first 30 minutes, charges will be billed in 15 minute increments.
MoFlo (only the facility operator is allowed to run the MoFlo)
- Analysis (generally only for >4 colors or UV laser use) – $150/hr
- Cell Sorting – $160 first hour, $130 each additional hour
- One hour minimum. After first 60 minutes, charges will be billed in 15 minute increments.
- Help in preparing figures for publication – $30/hr
Flow Cytometry Core Manager
Ms. Sarah Schuett, Manager