Frequently Asked Questions

General Questions

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

  1. Contact us to request an appointment or information:
    Sarah Neering: sjn16@psu.edu, for questions and training.
    Brian Dawson: bcd5018@psu.edu for questions, training and cell sorting appointments
  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 iLAB Instrument Managment System

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.

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. Please e-mail to inquire about external usage rates.

Current Internal Penn State rates:

  • BD LSR Fortessa: $54.00/hour
  • Amnis Flowsight: $75.00/hour
  • BC Astrios Cell Sorter: $106.00/hour plus an initial $75.00 setup fee
  • Miltenyi VYB: $50.00/hour
  • Miltenyi Tyto Sorter: $40.00/hour, sorting cartridges are $200.00 each

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.
  • It is highly recommended that users titrate their antibody reagents to determine the minimum amount of antibody required to get the maximum amount of fluorescence. Companies will very often recommend more than you need.

No Overbooking

  • Booking too much time inconveniences fellow researchers. Please be mindful of how long it takes you to run your samples so you can more accurately schedule future appointment lengths.
  • 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. Billing for training will include the equipment time, plus and additional staff training rate ($34.64/hr).

We currently do not train users on our cell sorters.

Required safety training

All users must complete Penn State EH&S Laser Safety training prior to being trained on any flow cytometer. You will need to bring a copy or e-mail staff a copy of the training certificate you receive at the end as well as e-mail a copy to ehslaser@psu.edu.

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, as well as which instrument you use.

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.

Cell Sorters

  • For cell sorting, concentration and quality of your sample are essential to running a sorting procedure. A dilute sample will take longer to go through, and an over-concentrated sample will take longer since we'll have to reduce the speed on the machine. For more on sample preparation and tips, please read our Sorting FAQ.
  • At full speed we can typically go through 1mL of sample in 20-30 minutes on the Astrios.

It's your data; you should take it with you and archive it in multiple places. The facility keeps temporary copies of flow cytometry data.

You are responsible for storing your data!

Data are routinely collected in listmode (all raw measurements are collected on each sample). Every 6 months, these listmode files are removed from the flow cytometer. Users can make download data files to BOX for analysis on any analysis package that supports FCS format (FlowJo software is available for free in the lab). NO USB PORTAL TRANSFER ALLOWED

Instrument Company Model Address
Flow Cytometer BD LSR Fortessa Franklin Lakes, NJ
Flow Cytometer
Miltenyi Biotec
MACS Quant Vyb
Bergisch Gladbach, Germany
Flow Cytometer
Amnis Flowsight Seattle, WA
Cell Sorter
Beckman Coulter
Astrios Moflo EQ
Brea, CA
Cell Sorter Miltenyi Biotec MACS Quant Tyto
Bergisch Gladbach, Germany

Safety issues

There are not many chemical hazards in the Flow Cytometry Core. However we still have chemicals like concentrated ethanol, bleach, and potentially toxic dyes like Propidium Iodide and DAPI, so gloves should be worn when working around any potential chemical or biological hazard in the lab. All samples run on our machines should be BSL-1 materials. If they are potentially infectious, or human cell lines they should be fixed before running them on any of our machines. The only exception is the Astrios cell sorter which can run up to BSL-2+ materials since it is housed in a bio-safety cabinet.

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. All users are required to complete laser safety training from EH&S and provide a copy of your certificate of completion to the Flow Cytometry staff as well as e-mailing a copy to ehslaser@psu.edu.

Policies

  • All fixatives, solvents and 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.

Other Questions

Getting Started

E-mail Brian Dawson (bcd5018@psu.edu) to schedule a new sorting appointment or for questions about sorting. For brand new experiments, often users will meet with staff to discuss the purpose and goal of the experiment and offer any advice for preparation or what settings on the machine may work best. For example: What kind of cells are you sorting? How many cells do you need? What concentration should the sample be? How much time is needed? Which fluorochromes?

Fill out the pre-sort form as best as you can. E-mail it or bring it with you for the discussion or before your sort. You only need to fill this out once unless the cell type or preparation procedure changes.

Cell Prep

Cell samples must be in a single-cell suspension. Clumps cause instability in the flow tip or can clog, causing a delay in continuing your sort and extending the length of your appointment. Stream turbulence will cause lower purities in your sorted tubes. We recommend a concentration around 5 million cells/mL. The sample can be suspended in the buffer that works best with your cell type to keep them alive during the sort. Bring extra buffer so the we can dilute the sample if it's too concentrated. (It's always easier to dilute rather than concentrate a sample.) A summary of our recommendations for sorting prep are here: Cell Sorting FAQ

Sort rates

12,000 and 25,000 events per second are the maximum sorting event rates for the 100um and 70um tips respectively. Please keep in mind that event rate is measuring every particle detected by the machine, including any debris not gated out. Your sorted population depends on the percentage of "good" cells in the selected populations. The actual speed of sorting your selected cells will depend on the percentage of positive cells and the concentration of the sample .

Please note that for smaller cell types like red blood cells, we recommend 60psi with the 70um tip. For larger cell types like cell lines we run the machine at 25psi on the 100um tip. This also reduces the risk of clogging.

Clumps

Cell samples must be filtered through 40 um nylon mesh to remove clumps of tissue, etc. Pour your sample through the mesh prior to sorting. We have nylon mesh filter tubes in the lab that we will use at cost ($1/tube) as needed if we notice any clumping in the tube you bring. 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.

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

Sample Tubes

Ideally, you can bring your single cell suspension in a Falcon #352063 12 x 75mm tube. We can run 1.5, 15 and 50mL tubes on the Astrios, but 5mL tubes are usually more effective at getting as much sample as possible through the machine.

Controls

You should ALWAYS bring an unstained control with your samples, along with single color preps for each different fluorochrome you use in your sorting sample. Bring them in Falcon tubes (see above). We can try to run your samples without them, but we cannot give any guarantee the gates are set correctly without a full set of controls to set voltages and gates.

Collection tubes

Sorted samples can be collected into 1.5mL micro-centrifuge tubes, 12x75mm 5mL FACS tubes, 15mL or 50 ml conical tubes. Bring the tubes of choice with you, containing some collection buffer to cushion the sorted cells (preferably something that your cells will be happy to be sorted into to help ensure as many survive the process as possible). It's best practice to bring a few 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. We can set the temperature anywhere between 5° and 40° C

Sterility

Completely sterile sorts are NOT possible. We run 10% bleach through the sheath and sample tubing after any potentially bio-hazardous or infectious samples are run, use sterile sheath fluid and sterilize the sort chamber ethanol. All preparation of the sample must be done as cleanly as possible and brought to the lab in a sterile fashion. The sample 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 8/2/2019

  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 schedule a sort. Call 814-863-2762 or email Brian (bcd5018@psu.edu) to schedule.
  3. NOTE: Time series experiments where multiple sorts will spaced out in set intervals must be approved and scheduled at least a week in advance to work around any potential conflicts in the schedule.

  4. 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 or email as soon as possible the morning of your sort to cancel with a staff member. Otherwise, you may be charged the set-up fee even if you do not come for the scheduled sort.

  5. If you are running late for your appointment, call 814-863-2762 or e-mail Brian (bcd5018@psu.edu) to give us an update on when you’ll be coming. Usage of the instrument will begin to be charged after 30 minutes from the originally scheduled start time if no notification has been given.

  6. 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 that we finish your sample by the end of the sorting day (4:30pm).

  7. 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 or researcher must contact Sarah Neering (sjn16@psu.edu) to discuss and get approval, before the staff member may perform the sorting.

A printable copy of this policy can be found here.


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

2019 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.

· Billing period is May 2019 May 2020 (295.90 $/year) for one license

FlowJo is available on three (3) desktop computers in W-124A Millennium Science Complex.

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 Sarah Neering (sjn16@psu.edu)