Flow Cytometry Facility

Frequently Asked Questions

General Questions

Our services are available to all Penn State departments on all campuses and to researchers outside the University.

The fee schedule for university affiliated researchers is subsidized by the Huck Institute for Life Sciences, which allows for much lower prices than the industry standard.

Non-university researchers cannot take advantage of the subsidized fee schedule.

  1. Contact us to request an appointment.
  2. Your first appointment will be a planning session
    • Future appointments will be scheduled at that time.
    • Be sure to bring along background literature illustrating the type of information you intend to collect.
  3. Register for a User account and add a billing account in the Research Instrumentation Management System (RIMS).

Billing rates vary according to the instrumentation. There is no charge for consultation on techniques, experimental design or subsequent data analysis.

Bills are generated each month electronically.

Estimate cost of experiments

  • Have students routinely estimate the cost of experiments... animals, reagents, antibodies, bench time, and facility time.
  • It is a great learning experience and useful for grant writing and running a lab in the future.
  • Carelessness does cost money.

No overbooking Pre Sort Form

  • Booking too much time inconveniences fellow researchers.
  • After several abuses we will begin to charge for this revenue-losing behavior.

It depends on what you want to do. We offer training on equipment use and sample preparation. Various University safety courses are also recommended or required.

Equipment use and sample preparation

We provide training on an individual basis. Training is usually divided into several 1-2 hour sessions each encompassing specimen preparation and basic equipment use.

Required safety training

If uranyl acetate is used in your protocol, you need to complete the Penn State radionucleotide safety training and exam.

Recommended safety training

All persons working in a laboratory should attend the chemical safety training seminar, a free course given by Environmental Health and Safety at Penn State.

It depends on the type and number of samples you want to analyze.

Benchtop cytometers

  • A working estimate is 1 minute per sample after setup (which usually takes around 30 minutes, depending on complexity).
  • 50 individual tubes (cell concentration 1million/ml) can easily be run in one hour.

It's your data; you should take it with you and archive it in multiple places. The facility keeps temporary copies depending on whether its flow cytometry or microscopy data.

You are responsible for storing your data!

Data are routinely collected in listmode (all raw measurements are collected on each cell). Once a month, these listmode files are removed from the flow cytometer and archived on a DVD. Users can make copies of these data files for analysis on any analysis package that supports FCS format (FCS ExpressFlowJo and CXP packages are available in the lab).

Instrument Company Model Address
TEM JEOL JEM 1200 EXII Peabody, MA
Bioscan Camera GATAN 792 Warrendale, PA
TEM High-resolution Camera Tietz F224 Gauting, Germany
SEM JEOL JSM 5400 Peabody, MA
SEM Backscatter Electron Imaging ETEP-Robinson RBJ-5400FML Sydney, Australia
Cryo-SEM GATAN C1500C Warrendale, PA
Image Archiving PGT IMX-PC v.10 Princeton, NJ
Electron Dispersive Spectroscopy PGT IMX-PC v.10 Princeton, NJ
Bioplex Bio-Rad 200 Hercules, CA
Flow Cytometer Beckman Coulter FC500 Brea, CA
Flow Cytometer Beckman Coulter XL- MCL Brea, CA
Flow Cytometer BD Fortessa Franklin Lakes, NJ
Sorter BD Influx Franklin Lakes, NJ
Paraffin Microtome Thermo Fisher Finesse ME Kalamazoo, MI
Paraffin microtome Leica 2040 Deerfield, IL
Ultramicrotome Leica UC6 Deefield, IL
Ultramicrotome Reichert Jung Ultracut E Deerfield, IL
Ultramicrotome Leica UCT Deerfield, IL
Cryo-ultramicrotome Leica FC6 Deerfield, IL
Diamond Knife Diatome Histo, ultra 45, etc Fort Washington, PA
Critical Point Dryer Techno Trade BALTEC CPD030 Manchester, NH
Sputter Coater Techno Trade BALTEC SCD050 Manchester, NH
Microwave Electron Microscopy Sciences 820 Fort Washington, PA
Cryostat Thermo Fisher 77210163 GB Kalamazoo, MI
Slide Stainer Thermo Fisher Varistain Gemini Kalamazoo, MI
Tissue Processor Thermo Fisher Citidel Kalamazoo, MI
Carbon Evaporator Boeckeler EMS950X Hatfield, PA
Automatic TEM Tissue Processor RMC EMP-5160 Tucson, AZ

Most likely you will have many citations from your own literature search. Some of the sources of information that we have used to develop general protocols are listed below.

  • Bell, P.B., 1984, Scanning Electron Microscopy of Cells in Culture, SEM, Inc. AMF O’Hare, IL.
  • Bozzola, John J. and Lonnie D. Russell 1999, Electron Microscopy: Principles and Techniques for Biologists, 2nd Ed., Sudbury, Ma.
  • Dykstra, M. J., 1992, Biological Electron Microscopy: Theory, Techniques and Troubleshooting, Plenaum Press, New York, NY.
  • Cross, P.C. and K.L. Mercer, 1993, Cell and Tissue Ultrastructure: A Functional Perspective. W.H. Freeman and Company, New York, NY.
  • Giberson, R.T. and Richard S. Demaree, 2001, Microwave Techniques and Protocols, Humana Press, Totowa, NJ.
  • Goldstein, J.I., D.E. Newbury, P. Echlin, D.C. Joy, C. Fiori, and E. Lifshin, 1981. SEM and X-Ray Microanalysis: A Text for Biologists, Material Scientists and Geologists, Plenum Press, New York, NY.
  • Hall, J.L. and C. Hawes, 1991, Electron Microscopy of Plant Cells, Academic Press, San Diego.
  • Hayat, M.A., 1989, Principles and Techniques of Electron Microscopy: Biological Applications, 3rd Ed., CRC Press, Inc. Boca Raton, FL.
  • Kok, L.P. and E. Boon. 1992. Microwave Cookbook for Microscopists, 3rd Ed. Coulomb Press, Leyden, Leiden, Netherlands.
  • Lee, R.L., 1993, Scanning Electron Microscopy and X-Ray Microanalysis, PTR Prentice-Hall Inc, Englewood Cliffs, NJ.
  • Rochow, T.G. and P.A. Tucker, Introduction to Microscopy by Means of Light, Electrons, X-Rays, or Acoustics, 2nd Ed., Plenum Press, New York, NY.
  • Rodin, J.A.G., 1977, Histology: A Text and Atlas, University Press, New York, NY.
  • Severs, N.J. and D.M. Shotton, Ed. 1995 Rapid Freezing, Freeze Fracture and Deep Etching: Techniques in Modern Biomedical Microscopy, Wiley-Liss, New York, NY.
  • Venable, J.H. and Coggeshall, R. 1965, A Simplified Lead Citrate Stain for Use in EM. , J Cell Biol, 25: 407-408.

Safety issues

Many of the chemicals used in electron microscopy protocols are hazardous. If handled improperly they may cause serious health problems. The purpose of raising this issue is not to scare or discourage anyone, but rather to educate users to properly and safely handle these chemicals. The regulations should be considered non-negotiable.

There is a zero tolerance policy for unsafe or careless handling of chemicals or equipment in the facility.

Recommendations

It is recommended that all persons working in a laboratory attend the chemical safety training seminar, a free course given by Environmental Health and Safety at Penn State.

Policies

  • All fixatives, solvents and embedding media must be handled in a hood with caution.
  • Assume all chemicals are hazardous, regardless of label, handle with caution.
  • Do not pour anything down the drain unless specifically directed to do so by a staff member.

More Resources

  • Smithwick, EB (1985) Cautions, Common Sense, and Rationale for the Electron Microscopy Laboratory. Journal of Electron Microscopy Technique 2: 193-200.
  • Barber, VC (1984) Safety in the Scanning Electron Microscopy Laboratory - 1984 Update. Scanning Electron Microscopy IV: 1719 - 1722

Are any radioactive materials used in the Facility?

Uranyl acetate is used in some of the staining procedures for TEM.  Uranyl acetate is radioactive, but when handled properly, does not pose a danger to the user.

How should I handle it?

If uranyl acetate is used in your protocol, you must use it with caution and dispose of it properly.  When working with radioactive materials, you must be careful not to contaminate other parts of the lab. There is a lead cabinet in the EM Facility for disposal of radioactive waste.

Required Training

Environmental Health and Safety requires all personnel working with radioactive materials (including uranyl acetate) to complete the radionucleotide safety training and exam.

Links

Books

The facility also maintains an extensive library and data base of existing protocols. We provide short-term loan of books.

  • Shapiro, H.M. (2003) Practical Flow Cytometry, Fourth Ed., John Wiley and Sons, Hoboken, NJ.
  • Hayat, M.A. (2000) Principles and Techniques of Electron Microscopy: Biological Applications, Fourth Ed., Cambridge University Press, Cambridge, UK
  • Bozzola, J.J. and Russell L.D. (1999) Electron Microscopy: Principles and Techniques for Biologists, 2nd Ed. Jones and Bartlett Publishers, Boston, MA.
  • Hall, J.L. and C. Hawes (1991) Electron Microscopy of Plant Cells, Academic Press, San Diego
  • Dykstra, M. J. (1992) Biological Electron Microscopy: Theory, Techniques and Troubleshooting, Plenaum Press, New York, NY.
  • Severs, N.J. and D.M. Shotton, Ed. (1995) Rapid Freezing, Freeze Fracture and Deep Etching: Techniques in Modern Biomedical Microscopy, Wiley-Liss, New York, NY
  • Rochow, T.G. and P.A. Tucker, Introduction to Microscopy by Means of Light, Electrons, X-Rays, or AcousticsSecond Ed., Plenum Press, New York, NY
  • Lee, R.L. (1993) Scanning Electron Microscopy and X-Ray Microanalysis, PTR Prentice-Hall Inc, Englewood Cliffs, NJ
  • Bell, P.B. (1984) Scanning Electron Microscopy of Cells in Culture SEM, Inc. AMF O'Hare, IL
  • Goldstein, J.I., D.E. Newbury, P. Echlin, D.C. Joy, C. Fiori, E. Lifshin (1981) SEM and X-Ray Microanalysis: A Text for Biologists, Material Scientists and Geologists, Plenum Press, New York, NY
  • Rodin, J.A.G. (1977) Histology: A Text and Atlas, Oxford University Press, New York, NY
  • Cross, P.C. and K.L. Mercer (1993) Cell and Tissue Ultrastructure: A Functional Perspective. W.H. Freeman and Company, New York, NY

Other Questions

Sign-up

Call the lab and talk to the sort operator to discuss your sort (you will be grilled). What kind of cells are you sorting? How many cells do you need? How much time is needed? Which fluorochromes? Fill out the pre-sort form. Email it or bring it with you for the discussion.

Cell prep

Pre-sort cells must be in a single-cell suspension. Clumps cause instability in the flow tip or can clog, causing frazzlement of the sorter operator. Stream turbulence will cause lower purities in your sorted tubes. We recommend a concentration of between 5 and 40 million per ml. The prep can be suspended in the cell's happiest buffer to keep them alive during the sort. Bring extra buffer for dilution of a too concentrated sample. (It's always easier to dilute rather than concentrate a sample.)

Sort rates

10,000 to 20,000 particles per second is a reasonable estimate for expected sorting rate. Remember that your sorted population depends on the percentage of "good" cells in the start population, as this table illustrates:

Sort rate (particles/s)Start concentration (particles/ml)Starting %Time to sort 106 cells (minutes)
10,0005 x 1061240
1035
5010
20,00010 x 1061180
1020
505

Or calculate likely rates for yourself using the tools at http://facs.scripps.edu/recovery.html.

Clumps

All cell samples must be filtered through 40 um nylon mesh to remove clumps of tissue, etc. We have nylon mesh filters in the lab. These can be sterilized by autoclaving. Pour your sample through the mesh prior to sorting. Rinsing with clear buffer will dilute your cell concentration, but help to avoid loss due to clogging. Filtering often causes loss of cells, but is unavoidable for a trouble-free sort. To determine how many cells are lost during a sort, count the cells after all antibody staining preparation and filtering, not just at the beginning of your prep.

Important Point: If filtering through mesh isn’t possible, be sure to call the lab and cancel your sort appointment.

Tubes

Bring your pre-sort single cell suspension in a Falcon #352063 12 x 75mm tube. This is a specific requirement—other tubes will not fit the sample head.

Controls

Always, always, always, every time, bring an unstained control, along with single color preps for each different fluorochrome you use in your sorting sample. Bring them in Falcon tubes (see above). The operator may refuse to sort if proper controls are not provided.

Collection tubes

Sorted samples can be collected into 1.5 microfuge tubes, 12x75 test tubes, or 15 or 50 ml conical centrifuge tubes. Bring the tubes of choice with you, containing a modicum of "happy" buffer to cushion the sorted cells. Always bring extra tubes. A clog will result in completely restarting the sort, discarding the contaminated collection tube. If sorting onto a plate (6, 12, 24, 96 well) only one population can be sorted at a time.

Temperature

It is possible to control the temperature of pre and post sort tubes. Please let us know in advance if this is necessary.

Sterility

Sterile sorts are indeed possible. We will run 70% EtOH or bleach through the sheath and sample tubing, use sterile sheath fluid and sterilize the sort chamber with UV light. You must remember that all preparation of the sample must be done sterility and brought to the lab in a sterile fashion and should include antibiotics in the media—both the sample and collection tubes. Control tubes should also be sterile preps. Collection tubes must be sterile with sterile media containing antibiotics. We cannot control what you bring into the lab. Remember most common bacterial contamination is caused by handling, not from the air.

Cell Sorting Policy – Updated on 11/2/2015

  1. Sorting can be primarily scheduled between the hours of 10:00am-4:30pm Monday-Friday. 
  2. A minimum of 24-hour notice must be given to cancel a sort.  Call 814-863-2762 to cancel.In case of an emergency/unexpected outcome (such as a loss of the research animal, cells dying, etc.) on the day of sorting, you must CALL 814-863-2762 by 9:00am to cancel with a staff member.  Otherwise, you will be charged an hour for set-up even if you do not come for the scheduled sort.
  3. If you are running late for your appointment, please CALL 814-863-2762 to tell the staff when you can come.  Usage of the instrument will begin to be charged after 30 minutes from the originally scheduled start time.
  4. If you are late, we cannot guarantee that your originally scheduled amount of time will still be available, since others also may have scheduled OR the end of the sorting day (4:30pm) may arrive. 
  5. Important Note: We will work together and try our best to accommodate your needs. However, if time outside of regular hours and days (10:00am-4:30pm Monday-Friday) is desired, or a request for the staff to continue sorting after 4:30pm due to the user's late arrival, the PI must contact Greg Ning (gxn7@psu.edu) to discuss the possibility before the staff member may perform the sorting.

The Huck Institutes maintains a volume license for the FlowJo software to provide a cost effective solution to researchers.

2015 FlowJo Licenses

FlowJo is an important data analysis software program for analyzing flow cytometry data.  Members of the Penn State research community may participate in the Huck Institutes’ site license for the FlowJo software, which provides a cost effective solution to researchers.  The following are the methods by which researchers at various levels may access the site license for 2015:

1.     Researchers from labs who paid for use of Facility flow cytometry equipment in 2014 are eligible to request complimentary license(s) in the following categories:

        a.     Frequent users of the Facility (transactions >$2k in 2014) will be provided complimentary license(s) upon request to be distributed on their lab’s computer(s).

        b.     A limited number of licenses for a 3-month period are available for less frequent Facility users upon request on a first-come, first-served basis.

               i.     For users planning to analyze a large number of files (>200) in one billing period, a complimentary license can be requested for use on a University-owned computer for one billing period at a time.

                               ·       Billing periods are

                                        o   February-April

                                        o   May-July

                                        o   August-October

                                        o   November-January

               ii.     If all licenses have been distributed for the requested billing period, users will be put on a waiting list for the next billing period. 

               iii.     Researchers who obtain a license but do not actually use it to analyze a large number of files (>200) will be prohibited from obtaining a license for the next 2 billing periods.

2.     For all researchers, including those from labs who did not use Facility flow cytometry equipment in 2014, complimentary access to FlowJo will be via:

        a.     FlowJo on two (2) desktop computers in W-124A Millennium Science Complex.

        b.     For researchers who require extended time for data analysis, a laptop computer (PC) with FlowJo versions 10 and 7.6.5, which can be signed out from the Facility for 1 week.

3.     Additional licenses may be purchased using the Facility’s discounted rate.  The anticipated rate for a 3-month billing period in 2015 is $69/computer ($276/year/computer).

pdf version of 2015 FlowJo Licenses

 Troubleshooting

  • There may be issues with your firewall or proxy server (if you have one). If so, please consult your network administrator to resolve the issue.
  • Please direct other questions or problems to Ruth Nissly.