|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
     |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
The Kimmel Cancer Center Flow Cytometry facility provides state-of-the-art fluorescence-activated cell sorting and analysis capabilities to investigators of the Kimmel Cancer Center and the Philadelphia region. The most commonly used applications are multi-color cell surface phenotyping, detection of intracellular cytokines and signaling molecules, transfection measurements, studies of apoptosis, cell cycle analysis, cell proliferation, and real time analysis of Ca2+ mobilization kinetics. The sorters, analyzers, and workstations allow for affordable, reliable, and accurate characterization of a wide variety of biological samples.
The services provided by the facility can be tailored to suit experimental designs in a variety of fields. We provide consultation on current protocols, analysis techniques, and data presentation. This resource plays a critical role in assisting KCC members performing various research projects directly related to the understanding of the biology and treatment of cancer. Furthermore the facility supports projects from investigators in Biochemistry, Genetics, Immunology, Medicine, Microbiology, Molecular Biology, Neurology, Pathology, Pharmacology, Surgery, and many other scientific fields.
Frequently Asked Questions:
Q. WHAT IS FLOW CYTOMETRY? A. Flow cytometry is a technique that measures properties of cells. It allows for qualification and quantification of cells and particles suspended in a fluid stream. Laser light of a single wavelength is directed onto this stream of fluid. Several detectors that are focused on the intersection of the laser and fluid stream collect the light signal generated. These detectors collect properties such as cell size, volume, granularity, and the presence of your parameter of interest.
Q. HOW DOES FLOW CYTOMETRY WORK? A. Here is a schematic of the basic principles of Flow Cytometry. The same principles apply to FACS, which will sort out live cells.
Q. WHAT CAN I MEASURE WITH FLOW CYTOMETRY? A.
Q. WHAT IS THE ADVANTAGE OF FLOW CYTOMETRY? A. The advantage of flow cytometry is rapid
simultaneous multi-parameter analysis.
It also allows for quantification of multiple cell phenotypes. The disadvantage is that you cannot
pinpoint where your molecule of interest is located within the cell as you can
with microscopy.
Q. HOW SHOULD I DESIGN MY FLOW CYTOMETRY EXPERIMENT FOR SUCCESS? A. First you'll need to decide which parameters you are interested in researching. Then you'll need to find that molecule attached to a fluorescent probe for that particular parameter. Here is a website that will help you locate your specific molecule of interest. Once you have located your probe you'll need to know if the lasers in the cytometer can excite your probe and detect the emission. You can always find protocols in the Current Protocols in Cytometry book in the facility. There are more resources for protocols such as the International Society for Advancement of Cytometry, the Purdue Cytometry website, and the Cytometry journal.
Q. WHAT ARE THE EXCITATION WAVELENGTHS OF THE CYTOMETERS IN THE FACILITY? A. The lasers in the facility cytometers have an excitation of 488 nm and 633 nm. Only fluorochromes excited at those wavelengths will work.
Q. WHAT ARE THE SUITABLE EMISSION WAVELENGTHS OF THE CYTOMETERS IN THE FACILITY? A. The most common wavelengths are 520nm, 570nm, 620 nm, and 670 nm. There are filters for many more colors but it's best to consult with the facility operator for designing a multicolor flow cytometry experiment. You can find a table listing fluorochrome excitations and emissions here. This link and this link will allow you to test your fluorochrome compatability. Once you have found a suitable fluorochromes for your multicolor experiment you should optimize the combination. Typically you should place less prevalent molecules on the brightest fluorochromes and vice versa.
Q. HOW CAN I GET TRAINED ON THE FLOW CYTOMETER? A. The best way to learn flow cytometry is to actually use the flow cytometer. Call the flow facility at extension 3.4556 or stop by room 606 in BLSB and set up an appointment with your friendly flow cytometry operator. In the mean time look at the web based training guide.
Q. HOW CAN I SIGN UP FOR AN APPOINTMENT TO PERFORM ANALYSIS OR CELL SORTING? A. After you finish the training session you can sign up with the on-line reservation system. If you need operator assistance for analysis please be sure to check the appropriate box. If you need a sorting appointment you have to call the operator to set up an appointment. Please call the facility at 215.503.4556 and be sure to give 48hr advance notice. You can always check the sort schedule on-line. *Please be advised that no shows or appointments cancelled without adequate notice will be billed at the normal rate and flow privileges may be revoked.
Q. WHAT DO I NEED TO SET UP THE CYTOMETER? A. As with all experiments you'll need the proper controls. In order to correctly set up the machine you'll need to remember a few things. All of your samples should be in 5 ml tubes. You'll need a negative control. The negative control will be unstained cells not your experimental negative. You'll also need a positive control for each fluorochrome you are considering. These are called compensation controls. For example, if you want to set up an experiment and use 3 fluorochromes you'll need 4 controls to properly set up the cytometer (1 negative and 3 compensation controls). For information on compensation you can look here. If compensation doesn't make sense you can always stop by the facility and talk to the flow operator. When you think you have an understanding of compensation try this Quiz.
Q. WHAT DO I NEED FOR CELL SORTING? A. Here are some good rules of thumb for cell sorting. You'll need to put your samples in 5 ml tubes. You'll need a negative control. The negative control will be unstained cells not your experimental negative. You'll also need a positive control for each fluorochrome you are considering. These are called compensation controls. For example, if you want to set up an experiment and use 5 fluorochromes you'll need 6 controls to properly set up the cytometer (1 negative and 5 compensation controls). For information on compensation you can look here. If compensation doesn't make sense you can always stop by the facility and talk to the flow operator. When you think you have an understanding of compensation try this Quiz. Additionally your sample should be sterile and the cells should be resuspened in either PBS or media with no more than 2% serum. You'll also need a collection tube with a few milliliters of media. The collection tube can be: an eppendorf tube, a 5ml tube, a 15ml tube, or a 50ml tube. The cell sorter has an additional function that allows it to sort into 6, 24, 48, and 96 well plates as well as custom slides.
Q. HOW MANY CELLS WILL I GET FROM CELL SORTING? A. You'll need to do a bit of math to get the answer. If you have 10 million cells and 50% are positive for your marker of interest then you'll receive 5 million sorted cells. The other 5 million cells are collected in the waste container. If you don't know how many cells you have and you don't know the percentage of cells you're interested in, then you don't know how many cells you'll get from sorting.
Q. HOW LONG WILL IT TAKE ME TO SORT? A. The cell sorter sorts at two speeds. Larger cells pass through a 100µm nozzle on the cell sorter at a rate of ≈12500 events/sec. This equates to 45x106 events/hr. Smaller cells are run at a higher pressure and speed on a 70µm nozzle. The rate for smaller cells is ≈22,000 events/sec and equates to 80 x106 events/hr.
Q. HOW MANY POPULATIONS CAN I SORT? A. You can simultaneously sort up to 4 separate populations.
Q. HOW MANY PARAMETERS CAN I QUANTIFY? A. Currently the high speed cell sorter can simultaneously measure 7 separate fluorochromes plus Forward and Side scatter. That makes 9 separate parameters you can measure! The analyzers can accommodate 4 fluorochromes each.
Q. WHAT NEW TECHNOLOGIES EXIST IN FLOW CYTOMETRY? A. The MoFlo cell sorter allows for single cell sorting into 96 well plates for cloning purposes. Furthermore, the Kimmel Cancer Center Flow Cytometry facility is now certified with a >90% success rate of placing single cells onto the Advalytix AmpliGrid PCR slide. The AmpliGrid slides are used in subsequent PCR or RT-PCR reactions on the AmpliSpeed slide cycler. We recommend AmpliGrid for single cell molecular analysis because you can visually verify correct cell placement on the slide and the analysis is more sensitive in the 1 ul reaction volume.
Q. WHAT INSTRUMENTS DO YOU HAVE IN THE LAB? A.
Q. HOW CAN I ANALYZE MY DATA? A. You can always analyze your data with the software installed on the cytometers. You can use Cell Quest on the FACSCalibur, System II on the Epics XL, and Summit on the MoFlo sorter. If you want a free analysis software for your laboratory PC you can download WinMDI. The University also has a site license for FlowJo flow cytometry analysis software. FlowJo is also installed on the workstation in the Flow Facility located in BLSB Room 606.
Q. WHAT DATA HAS BEEN RECENTLY PUBLISHED FROM THIS FACILITY? A. Multiple publications have been accepted in journals with the data and results generated by the analyzers and sorters in the facility. The following selections are figures from those publications. Typical applications include multiparamter analysis such as that in Figure 1. In this instance, cells were analyzed using the Coulter EPICS XL-MCL and the figure was generated with FlowJo software. The results show B cell proliferation during an immune response using CFSE labelling. A variety of kinetics assays can also be performed such as the calcium influx experiment shown in Figure 2. Cells were analyzed using the BD FACSCalibur and the figure was generated with FlowJo software. Results show calcium influx on CD16 triggered NK cells with and without Prostaglandin treatment. The data in Figure 3 displays a typical two fluorochrome immuno-stain performed on the Coulter Epics XL-MCL. Mouse bone marrow cells and splenocytes were analyzed for levels of Gr-1myeloid differentiation markers and transcription factors for granulocyte differentiation. The GFP positive cells were sorted on the Dako MoFlo sorter and a subsequent cell cycle anaylsis was performed by propidium iodide staining. Both the proliferation and differentiation capabilities of these mouse splenocytes and hematopoietic cells were assessed.
Citations and recent publications:
Alabyev B, Rahman ZS, and Manser T. J Immunol. 2007 May 1;178(9):5623-34 Alabyev B, Vuyyuru R, Manser T. Int Immunol. 2008 Oct; 20(10):1279-87. Chen Y, Perussia B, Campbell KS. J Immunol. 2007 Sep 1;179(5):2766-73. Fernandes-Alnemri T, Alnemri ES. Methods Enzymol. 2008;442:251-70.
|
