The Penn State Hershey Flow Cytometry Core Facility provides sophisticated fluorescence-activated cell sorting and analytical services at reasonable hourly rates. The facility is operated to provide access to both clinical labs and basic science investigators.
As a clinically certified facility, the core lab performs clinical analyses of patient samples on a regular basis.
In addition, five flow cytometers are dedicated to research and available for use by investigators at the College of Medicine:
• 4-color FACSCalibur
• 8-color FACSCanto
• 14-color LSR Fortessa
• 15-color LSR II SORP
• 16-color FACSAria SORP high speed cell sorter
Please take a few minutes to look over the website pages to ensure your flow cytometry experiments are conducted using appropriate methods and techniques to produce the most accurate data in a cost efficient manner.
- Core Policies & Procedures
- Safety Guidelines
- Sample Requirements and Protocols
- Cell Sorting Requirements
- Genologics Lablink
- Off Hours Usage
- Flow Cytometry Basics
- Helpful Resources
- Recent Publications
- Contact Us
Rates for Flow Cytometer Use (Updated March 1, 2010)
Standard Sample Acquisition (Researcher provides operator)
Standard Data Analysis (Researcher provides operator)
Assisted Acquisition or Data Analysis (Core provides operator)
Standard Sorting on FACSAria (Researcher provides assistant)
Sorting Setup Charge
Special Sorter Setup Charge
Minimum Charge for Sorting Use
NOTE: "No Shows" and cancellations less than 8 hours in advance may be charged for scheduled time.
General Procedures for the Core
The Flow Cytometry Core Facility serves both the Hospital and the Researchers of the College of Medicine. There are three instruments (FACSCanto and 2 FACSCaliburs) dedicated to clinical use. Use of these machines by researchers are prohibited unless specifically directed to do so by the Core personnel. All other instruments, including the Cell Sorter, are for research use and must be scheduled in advance for use (see above).
Billing for usage is done monthly according to Department and Principal Investigator. We do not break down the billing by technician or project, but if the researcher needs this information, they can retrieve it upon request to the core facility. All users must fill out the Proteus LABLINK page information each time they use equipment.
The facility is designed to have the users operate the instruments when running samples and analyzing data. The Core personnel will train these users to become proficient over time in setting up their experiments, running samples and producing and archiving data resulting from the experiments.
There is a terabyte server available to all the researchers to use for transferring data from the DIVA workstations to your laboratory for archival. The server space (\\hersheymed.net\Files\research\corefacility\Results\Flow Cytometry) is to be used only as a dropbox for later transfer to your own archival system. Users may also transfer data to other portable storage media if desired. In any case, the user is ultimately responsible for archiving any and all data generated. The DIVA workstations will only retain up to 3 months of your work in the DIVA database and Export folders. The research server is not a secure space to archive data so again, the user is responsible to archive flow cytometry data, not the core lab.
Safety for all users and Core personnel is a priority concern. Since the Core facility is a shared laboratory and is designated as a BSL 2 facility, there are certain guidelines that all users are responsible to be aware of and follow. This is a special concern for cell sorting and biosafety concerns and guidelines will be strictly adhered to. This policy is summarized in the Safety Guidelines. They include guidelines for the safe use of lasers as well.
Summary of Guidelines for the Safe Use of the Flow Cytometry Facility
The Flow Cytometry Facility operates as a Biosafety Level 2 laboratory. These guidelines are reviewed and revised if necessary each year and each facility user is responsible to review them on a regular basis to insure they are in compliance. Occasionally, some cell sorting experiments may operate in the cell sorter room under BSL-2+ conditions. This will be determined by the Flow Core Staff.
1) Admission to the laboratory is restricted to authorized personnel when work with human samples or a known infectious agent is in progress.
2) All cells must be assigned appropriate BSL levels for research use by the Biological Safety and Recombinant DNA Committee (Ralph Keil, Ph.D., Chair) before they are allowed to be used in the Flow Cytometry Core. Additional information can be found on the Safety Infonet website.
3) No BSL2+ samples are allowed to be run on the analyzers.
4) Before any cell sorting experiment can be scheduled, the Cell Sorting Experiment Form must be completed, signed and presented to the operator. This allows time for appropriate evaluation of biohazards under high speed sorting conditions. This must be done at least a week before the proposed experiment.
5) Some unfixed, primary human and primate cells and other BSL2 cells to be used on the FACSAria High Speed Cell Sorter may be designated as BSL2+ by the Flow Biosafety Committee. In these cases, strict adherence to the BSL2+ SOP will be required (located in the Flow Core). These same cells MAY be used under BSL2 conditions on the Cell Sorter if they are fixed according to accepted methods of inactivating all biohazards. (See note on Special Requirement for Sorting Primary Human Cells below.)
6) Whenever possible, samples of human or primate origin to be run on any cytometer should be fixed according to a process known and documented to inactivate HIV and other biohazards.
7) All procedures must be performed carefully to minimize the creation of aerosols.
8) All samples and time used on the instruments must be logged into the facility in Proteus. Samples that have been fixed for analyzing or sorting on the Cell Sorter may be monitored and recorded to insure that the fixation is adequate.
9) Work surfaces are to be decontaminated with 10% household bleach (FACS Clean) after any spillage of sample or hazardous material and after each user completes his/her work.
10) Instrument decontamination shall be performed according to protocols posted on each instrument.
11) All spills, however small, must be recorded in the accident book and reported to the Lab Manager.
12) All liquid and solid waste and disposal is to be removed from the laboratory as biohazardous material by each user. This includes all nuclear staining reagents such as PI, EB, AO, etc.
13) Gloves must be worn whenever handling or running biohazardous samples on the cytometer.
14) Hands must be washed after handling biohazardous materials and before leaving the laboratory.
15) Laboratory coats must be worn in the work area. These coats should be removed before leaving the laboratory.
16) Neither syringes nor hypodermic needles should be used in the facility without the consent of the Laboratory Manager. Glass objects should only be used when absolutely necessary.
17) No eating, drinking, smoking or applying cosmetics in the work area.
18) No mouth pipetting.
19) No animals are permitted within the laboratory.
20) All biohazard samples must be transported to and from the Flow Core in double sealed containers per College of Medicine safety policy.
In addition to the above guidelines and because this facility uses Class 3a through Class 4 lasers, the following Laser Safety guidelines shall also be observed:
- Access to the laboratory will be limited to only those persons necessary to the running of experiments when any laser is operating in the UV mode or when it is necessary to operate a laser without protective shielding.
- In such cases, the appropriate protective goggles or glasses will be made available and must be worn by those in the work area.
- Only qualified personnel shall operate or adjust any laser.
In the event of a potential retrovirus exposure
This item refers to exposure of skin or mucous membranes to infectious materials, and is modeled after the NIH program, "3 Emergency Steps to Take in the Event of a Potential HIV Exposure". Remember that the risk of infection is very dependent upon the titer of virus and the route of exposure. Thus the risk of infection by contact of intact skin with infectious body fluids is probably truly zero, and although the risk of infection by contact of mucous membranes or non-intact skin with infectious body fluids may be extremely small, it is likely to be no-zero.
The 3 emergency steps are:
- Immediately initiate first aid at the work site.
- Contaminated skin should be meticulously cleaned for 10 minutes using a povidone iodine solution (such as Betadine) and copious amounts of water.
- Contaminated eyes and mucous membranes should be irrigated for 5 minutes using water.
- Notify your supervisor, if he or she is immediately available. More importantly, go on to step 3.
- Report to the hospital Emergency Room to activate the invasive accident protocol (needlestick protocol), which will include evaluation, counseling, and provision of antiretroviral treatment if deemed appropriate. Do this immediately (i.e. within the half-hour).
General Sample Requirements
TUBES: Cells must be brought in Falcon 12x75mm polystyrene test tubes (Falcon # 2008) for running on any of the analyzers. Other tubes of varying sizes may be used on the cell sorter. Collection tubes for cell sorting should be polypropylene.
VOLUMES and CONCENTRATIONS: Minimum sample volumes per tube are .5 ml. Cell concentrations should be .5 - 1.0 X 106 cells per ml.
FLUOROCHROME SELECTION: Please review the capabilities of the instruments before selecting your fluorochromes and dyes to ensure that the instruments can excite and detect the dyes of choice. For multicolor experiments, it is imperative that you consult fluorochrome guidelines or personnel in the facility for best possible outcomes.
COMPENSATION CONTROLS: Bring positive and negative controls. A negative control is an unstained sample of cells in PBS or fixative or a sample of cells stained only with the secondary ab if this is the system you are using. If you have samples stained with more than one fluorochrome per test tube, bring in a sample stained with each fluorochrome individually. This is for compensation purposes. If you do not have enough positive stained cells for each fluorochrome, you may use antibody capture beads. To make conclusions about your experiment, the proper controls are critical. If you have any questions as to what the appropriate controls are, do not hesitate to call facility personnel at ext. 6908.
General Cell Staining Protocols
Cell Surface Marker/Antigen Staining
For each control and test sample, prepare a single cell suspension of 1X106 cells in cold PBS. Wash cells 1X in at least 1 ml of PBS with 2% FBS/media, centrifuge cells and aspirate supernatant.
Resuspend cells in a final volume of 50ul containing the primary antibody at the correct titer and PBS with 2% FBS/media. Reference manufacturer's guidelines but in general, incubate on ice or at room temp. for 20-30 minutes.
Wash cells 2X in PBS with 2% FBS/media. Centrifuge and aspirate supernatant.
For a directly conjugated antibody go to the next step. For indirect labeling, resuspend cell pellet in a final volume of 50ul containing the Secondary antibody and PBS with 2% FBS/media.
Resuspend cells at a final concentration of 1-2X106 cells/ml in at least 0.4ml fresh 1-2% formaldehyde/PBS. Provide the proper controls and analyze by flow cytometry.
Intracellular Staining- there are also good commercial kits available
- PI Staining of ETOH fixed cells for DNA Content, Cell Cycle and Apoptosis (Easiest Method)
- PI Staining of Nuclei for DNA Content and Cell Cycle (Best Resolution)
- There are good commercial kits available. We recommend using kits with Annexin V PE and 7AAD as the best fluorochrome combination.
Current Protocols in Cytometry is available in the Flow Core upon request.
For information on other protocols, visit some of our other links or consult with resources in the Flow Core Facility.
Cell Sorting Requirements
Cells should be concentrated to at least 5 million cells/ml. If you want to sort lots of cells in a short amount of time, you may concentrate your cells even more (but the more concentrated your cells are, the more likely they might form clumps). Remember to calculate how many total cells you will need to analyze in order to sort the required number of target cells you want to have after the sort experiment. In general, lymphocyte sized cells can be run at rates close to 30K/sec. and cell lines can be run around 12K/sec.
You will need to bring cell media in the tubes or trays into which you want to sort cells. Tubes should be polypropylene to avoid static charges and can vary in size. Media should fill about 1/3 of the tube space before collection begins.
It is required that you filter your cells before sorting. Clogs will be a problem to any sorting experiment and increase biohazard risks. 35 or 40um filter cap tubes are available from BD Biosciences for this purpose.
Also, you must read and adhere to the SAFETY GUIDELINES before sorting unfixed cells and you must fill out and have the P.I. sign the CELL SORTING EXPERIMENT form before you can schedule any sorting experiment. This form must be given to the core personnel for review ONE WEEK prior to scheduling your sorting experiment.
If, after our review of the CELL SORTING EXPERIMENT form, it is determined that your experiment is at the BSL2+ level, you will be required to read and adhere to the Flow Cytometry BLS2+ SOP for cell sorting which is available in the cell sorter room.
Special Requirement for Sorting Primary Human Cells
Any request to sort live, primary human cells in the Flow Cytometry Core will require a review of the samples in question by the Flow Core Director based on the information on the CELL SORTING EXPERIMENT form and possible consultation with the PI involved. (This does not necessarily apply to human cells that are from an established published cell line.)
Several outcomes of this evaluation are possible. Some high risk populations may be deemed too high-risk to sort in our facility. Other populations may be sorted under BSL2 conditions, others under BSL2+ conditions and still others only after the donors are tested for HIV antibody and Hepatitis C and B Core antibody.
If donor testing is required, you must contact the Clinical Trials Coordinator for more information on this process. Once the tests are completed, a copy of the test report without patient ID will be signed by the requesting PI and forwarded to the Flow Cytometry Facility before the cell sorting experiment is performed.
Scheduling for All Flow Cytometers
Scheduling time for sample acquisition or data analysis is done by calling the Core facility at Ext. 6908. Researchers are scheduled on a first come first served basis. A SCHEDULING CALENDAR has been made available for all users to view online to plan experiments. Please "bookmark" the link for future use. It is "view only" and will change often so be sure to refresh the site as you make your plans. Scheduling is best done a few days in advance to ensure that you have sufficient time and that you will be able to run your samples within a reasonable time after they have been prepared. If you are doing a time course study, it is best to check with the facility well in advance to ensure that someone will be there on the days you need to have access to the cytometers. Occasionally a request for live cell analysis will take priority over a previously scheduled time slot using fixed cells.
Requests for cell sorting must be made at least a week in advance to ensure assistance will be available and that all biosafety concerns can be addressed.
Click the link above to access the Genologics Lablink site.
Use of Facility During Off Hours
Because of the nature of certain research projects it is sometimes necessary that accommodations be made for the processing of samples through the flow cytometers in the absence of the Flow Cytometry Core personnel who normally set up the instruments and assist the users for data acquisition. The Flow Cytometry Core has a Specialist and a trained backup Research Associate to cover almost all of the normal business hours for research use. However, there are rare occasions when neither person will be available to assist users even during regular business hours. For this reason, it is necessary to consult the Scheduling Calendar well in advance to ensure that your project can be scheduled accordingly.
For times other than regular business hours (i.e. evenings or weekends), certain approved users will also be able to use the flow cytometers upon prior arrangement with the Core personnel. There is a key pad lock on the door but the code will be made available for these users. These users will be completely "on their own" in starting up the instrumentation and shutting it down properly.
In the event that problems would arise, the operator has the option of calling the Lab Manager, Nate Sheaffer, at (717) 571-6231 (cell phone). If he can not be reached or the problem can not be solved over the phone, it will be necessary to stop the processing of samples at that point and wait until Core personnel return to the Lab. These special arrangements need to be scheduled ahead of time with the approval of the Core personnel.
To be approved for such "off hours" data acquisition, please see one of the Core personnel to complete the necessary training checklist for the particular instrument that will be used. For any occasion to process samples when Core personnel are not in the lab, only the Research FACSCalibur, FACSCanto II, LSR II and LSR Fortessa are available for research use. There are special arrangements for the other instruments to be made available for the processing of clinical specimens.
The FACSAria Cell Sorter will not be available for use in the absence of Core personnel at any time.
Laser excitation wavelengths (2):
Blue 488; Red 635nm
Fluorescence detection (4): 530, 585, >650; >670
BD FACSCanto II
Laser excitation wavelengths (3):
Blue 488; Red 635nm; Violet 405nm
Fluorescence detection (8):
BD FACSAria SORP
Laser excitation wavelengths (4):
Blue 488; Red 635; Violet 405nm; Green 532nm
Fluorescence detection (16):
Users are expected to accompany their samples and assist in the monitoring of data acquisition and sorting experiments. The Core personnel will be responsible for all the set up and shut down procedures related to each experiment. For more information on the policies for using the FACSAria please see the Scheduling/Fees/General Procedures and the General Sample Requirements and Protocols. The cell sorting experiment report must be submitted prior to scheduling a sort on the FACSAria.
Special Order BD LSR II
Laser excitation wavelengths (4):
Blue 488; Red 635; Green 532; Violet 405nm
Fluorescence detection (15):
BD LSR Fortessa
Laser excitation wavelengths (3):
Blue 488; Red 640; Violet 405nm
Fluorescence detection (14):
Flow Cytometry Basics
Flow cytometry is a means of identifying and measuring certain physical and chemical characteristics of cells or particles as they travel in suspension one by one past a sensing point. The flow cytometer is able to "look" at thousands of cells or particles per second and perform and record many simultaneous measurements for each cell or particle.
The flow cytometer consists of a light source, collection optics, electronics and a computer to translate signals to data. The light source of choice is usually a laser which emits coherent light at a specified wavelength. Scattered and emitted fluorescent light is collected by two lenses (one set in front of the light source and one set at right angles) and by a series of optics, beam splitters and filters, specific bands of fluorescence can be measured.
A flow cytometer can measure physical characteristics such as cell size, shape and internal complexity and, of course, any cell component or function that can be detected by a marker attached to a fluorescent compound can be examined. A number of these cell markers and measurements can be made for each cell and combined to give an informative summary of the characterization, identification and function of large populations of cells. So the applications of flow cytometry are numerous, and this has led to the widespread use of these instruments in the biological and medical fields. See Helpful Resources for more information and introductions to Flow Cytometry.
Diagram courtesy of DakoCytomation
Some of the more common research applications include: immunology, cell cycle and cell growth, cell function and activation, cell differentiation, apoptosis, platelet activation, toxicology and Green Fluorescent protein detection. Some of the common clinical studies include: leukemia and lymphoma characterization, immune studies such as T and B cell subset determinations, stem cell content monitoring for transplant, nuclear ploidy and cell cycle determinations and reticulocyte counting.
In addition to analyzing populations of cells and particles for information and data which can be stored electronically and displayed in the form of dot plots and histograms, some flow cytometers have the ability to physically sort out cells or particles of interest. These cells can be sorted out of a heterogeneous mixture into a very pure population for further studies.
Introductions to Flow Cytometry
- Video Introduction to Flow Cytometry
- BD Biosciences Introduction to Flow Cytometry
- Other Flow Cytometry Tutorials
- Free Online Book: "Flow Cytometry - A Basic Introduction" by Michael Ormerod
Fluorochrome Choices and Controls
- Core Facility Canto II Fluorochrome Chart
- Core Facility Fortessa Fluorochrome Chart
- Core Facility LSR II Fluorochrome Chart
- Core Facility FACSAria Fluorochrome Chart
- Generic Fluorescence Spectra Viewer
- BD Biosciences Fluorochrome Guides and Spectra Viewer
- Another Fluorochrome Table
- Protocols from BD Biosciences
- Current Protocols in Cytometry (see lab manager)
- Purdue University Cytometry (Very Comprehensive Resource)
- International Society for the Advancement of Cytometry
- Catalog of Free Flow Cytometry Software
- BD Biosciences (FACSCalibur, FACSCanto, LSR II, FACSAria)
- FlowJo Software Manual
- Molecular Probes (Invitrogen)
Other Related Resources
Some recent publications which used data generated in the Flow Cytometry Core are listed below
Direct Presentation Regulates the Magnitude of the CD8+ T Cell Response To Cell-Associated Antigen through Prolonged T Cell Proliferation (2010)
Angela M. Tatum1, Alan M. WatsonTodd D. Schell article
A Marked Reduction in Priming of Cytotoxic CD8 + T Cells Mediated by Stress-Induced Glucocorticoids Involves Multiple Deficiencies in Cross-Presentation by Dendritic Cells (2010)
John T. Hunzeker, Michael D. Elftman, Jennifer C. Mellinger, Michael F. Princiotta, Robert H. Bonneau, Mary E. Truckenmiller and Christopher C. Norbury doi: article
Renal Dendritic Cells Ameliorate Nephrotoxic Acute Kidney Injury (2010)
Raghu K. Tadagavadi* and W. Brian Reeves article
Enhanced Osteoclastic Resorption and Responsiveness to Mechanical Load in Gap Junction Deficient Bone (2011)
Yue Zhang, Emmanuel M. Paul, Vikram Sathyendra, Andrew Davison, Neil Sharkey, Sarah Bronson, Sundar Srinivasan, Ted S. Gross, Henry J. Donahue article
PRAS40 Regulates Protein Synthesis and Cell Cycle in C2C12 Myoblasts (2010)
Abid A Kazi and Charles H Lang article
Therapeutic efficacy of FTY720 in a rat model of natural killer cell leukemia (2011)
Aijun Liao, Kathleen Broeg, Todd Fox, Su-Fern Tan, Rebecca Watters, Mithun Vinod Shah, Lucy Q. Zhang, Yongping Li, Lindsay Ryland, Jun Yang, Cesar Aliaga, Alden Dewey, Andrew Rogers, Kelly Loughran, Leah Hirsch, Nancy Ruth Jarbadan, Kendall Thomas Baab, Jason Liao, Hong-Gang Wang, Mark Kester, Dhimant Desai, Shantu Amin, Thomas P. Loughran, Jr. and Xin Liu article
A non-coding cationic lipid DNA complex produces lasting anti-leukemic effects (2010)
Nikki Keasey, Zachary Herse, Stella Chang, Denny H. Liggitt, Marla Lay, Jeffery Fairman and David F. Claxton article
Regulation of natural killer T-cell development by deubiquitinase CYLD (2010)
Andrew J Lee1, Xiaofei Zhou, Mikyoung Chang, John Hunzeker, Robert H Bonneau, Dapeng Zhou and Shao-Cong Sun article
Dendritic Cell Migration Limits the Duration of CD8 + T-Cell Priming to Peripheral Viral Antigen (2010)
Amanda M. Schell, Erica L. Granger, Frank Koczot, Matthew A. Fischer and Christopher C. Norbury article
FoxP3aand Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development (2010)
Rizwanul Haque, Fengyang Lei, Xiaofang Xiong, Yuzhang Wu and Jianxun Song article
Endogenous IL-10 attenuates cisplatin nephrotoxicity: role of dendritic cells (2011)
Raghu Kempegowda Tadagavadi and William Brian Reeves article
CD11b+, Ly6G+ Cells Produce Type I Interferon andExhibit Tissue Protective Properties Following Peripheral Virus Infection (2011)
Matthew A. Fischer, Michael L. Davies, Irene E. Reider, Erica L. Heipertz, Melanie R. Epler, Janet J. Sei, Molly A. Ingersoll, Nico Van Rooijen, Gwendalyn J. Randolph, Christopher C. Norbury article
Primary cell lines: false representation or model system? a comparison of four human colorectal tumors and their coordinately established cell lines (2010)
Danielle M. Pastor, Lisa S. Poritz, Thomas L. Olson, Christina L. Kline, Leonard R. Harris III, Walter A. Koltun, Vernon M. Chinchilli, Rosalyn B. Irby article
Read-through Activation of Transcription in a Cellular Genomic Context (2010)
Li Shen, David J. Spector article
Systemic Isotretinoin Therapy Normalizes Exaggerated TLR-2-Mediated Innate Immune Responses in Acne Patients (2012)
Melanie C. Dispenza, Ellen B. Wolpert, Kathryn L. Gilliland, Jenny P. Dai, Zhaoyuan Cong,
Amanda M. Nelson and Diane M. Thiboutot article preview
Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia (2010)
Xin Liu, Lindsay Ryland, Jun Yang, Aijun Liao, Cesar Aliaga, Rebecca Watts, Su-Fern Tan, James Kaiser, Sriram S. Shanmugavelandy, Andrew Rogers, Kathleen Loughran, Bailey Petersen, Jonathan Yuen, Fanxue Meng, Kendall Thomas Baab, Nancy Ruth Jarbadan, Kathleen Broeg, Ranran Zhang, Jason Liao, Thomas Joseph Sayers, Mark Kester and Thomas P. Loughran, Jr article
If you have any other articles which used data from the Flow Cytometry Facility, please contact Nate Sheaffer at email@example.com.
Todd Schell, PhD
Director, Flow Cytometry Core
Phone (717) 531-8169
Flow Cytometry Core Advisory Panel
- Bruce Stanley, Ph.D., Director of Research Resources
- Todd Schell, Ph.D., Director of Flow Cytometry Core
- Nate Sheaffer, Core Lab Manager, Flow Cytometry Specialist
- Christopher Norbury, Ph.D., Research Cytometry and Biosafety Advisor
- Jeff Sample, Ph.D., Biosafety Advisor
- Mike Bayerl, M.D., Clinical Cytometry Advisor
The daily operations as well as the review of clinical samples are the responsibility of:
| Nate Sheaffer, BA, ASCP Cyt |
Core Lab Manager
Phone (717) 531-6908
Joseph Bednarczyk, BS
| Jade Vogel, BS |
Phone (717) 531-6908