Flow Cytomtery and Cell Sorting

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.

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.

Laser excitation wavelengths

Fluorescence detection

Laser excitation wavelengths (three)

• Blue: 488nm

• Red: 635nm

• Violet: 405nm

Fluorescence detection (10)

• Blue: 530nm, 575nm, 695nm, 780nm

• Red: 670nm, 712nm, 780nm

• Violet: 450nm, 525nm, 605nm

BD-recommended color combinations:

• Six-color: FITC, PE, PERCP-CY5.5, PE-CY7, APC, APC-CY7

• Eight-color: FITC, PE, PERCP-CY5.5, PE-CY7, APC, APC-CY7, AMCYAN or V500, V450

Note: Alexa dye numbers are named using excitation wavelength whereas E Fluor and BD V dyes are named using emission wavelengths.

Revised: March 14, 2016

Laser excitation wavelengths (four)

• Blue: 488nm

• Red: 640nm

• Green: 532nm

• Violet: 405nm

Fluorescence detection (16)

• Blue: 515nm, 695nm

• Red: 670nm, 730nm, 780nm

• Green: 585nm, 610nm, 670nm, 710nm, 780nm

• Violet: 450nm, 525nm, 605nm, 655nm, 710nm, 780nm

BD-recommended color combinations:

• Six-color: FITC, PE, PERCP-CY5.5, PE-CY7, APC, APC-CY7

• Eight-color: FITC, PE, PERCP-CY5.5, PE-CY7, APC, APC-CY7, BV 421, BV 711

• 10-color: FITC, PE, PE-Texas Red, PERCP-CY5.5, PE-CY7, APC, Alexa 680 or 700, APC-CY7, BV 421, BV 711

Note: Alexa dye numbers are named using excitation wavelength whereas E Fluor and BD V dyes are named using emission wavelengths.

Revised: Oct. 27, 2017

Laser excitation wavelengths (four)

• Blue: 488nm

• Red: 635nm

• Violet: 405nm

• Green: 532nm

Fluorescence detection (16)

• Blue: 525nm, 582nm, 710nm

• Red: 670nm, 730nm, 780nm

• Green: 575nm, 610nm, 670nm, 710nm, 780nm

• Violet: 450nm, 525nm, 610nm, 670nm, 710nm, 780nm

Using this equipment

Users are expected to accompany their samples and assist in the monitoring of data acquisition and sorting experiments. Core personnel will be responsible for all the setup and shutdown procedures related to each experiment.

The cell sorting experiment form must be submitted prior to scheduling a sort on the FACSAria.

These choices are arranged in the same order that the FACSAria software uses.

BD-recommended color combinations:

• Six-color: FITC, PE, PERCP-CY5.5, PE-CY7, APC, APC-CY7

• Eight-color: FITC, PE, PERCP-CY5.5, PE-CY7, APC, APC-CY7, AMCYAN or V500, V450

• 10-color: FITC, PE, PE-Texas Red, PERCP-CY5.5, PE-CY7, APC, Alexa 680 or 700, APC-CY7, AMCYAN or V500, V450

Note: Alexa dye numbers are named using excitation wavelength whereas E Fluor and BD V dyes are named using emission wavelengths.

Revised: Oct. 27, 2017

Laser excitation wavelengths (four)

• Blue 488nm

• Violet 405nm

• Red 637nm

• Yellow Green 561nm

Fluorescence detection (17)

• Blue: 515nm, 610nm, 710nm, 780nm

• Violet: 431nm, 470nm, 610nm, 670nm, 710nm, 780nm

• Red: 670nm, 710nm, 780nm

• Yellow Green: 586nm, 610nm, 670nm, 780nm

Note: Alexa dye numbers are named using excitation wavelength whereas E Fluor and BD V dyes are named using emission wavelengths.

Revised: Feb. 7, 2020

Laser excitation wavelengths (five)

• Blue 488nm

• Violet 405nm

• Red 637nm

• Ultra Violet 355nm

• Yellow Green 561nm

Fluorescence detection (23)

• Blue: 515nm, 610nm, 710nm, 780nm

• Violet: 431nm, 470nm, 610nm, 670nm, 710nm, 780nm

• Red: 670nm, 710nm, 780nm

• Ultra Violet: 378nm, 515nm, 586nm, 670nm, 740nm, 820nm

• Yellow Green: 586nm, 610nm, 670nm, 780nm

Note: Alexa dye numbers are named using excitation wavelength whereas E Fluor and BD V dyes are named using emission wavelengths.

Revised: Feb. 7, 2020

Laser excitation wavelengths (three)

• Blue 488nm

• Red 640nm

• Violet 405nm

Fluorescence detection (nine)

• Blue: 527nm, 586nm, 700nm, 783nm

• Red: 660nm, 783nm

• Violet: 448nm, 582nm, >765nm

Note: Alexa dye numbers are named using excitation wavelength whereas E Fluor and BD V dyes are named using emission wavelengths.

BD FACSDiscover S8 Spectral sorter w Cellview Image Technology

For single cell analysis

This table lists the Band Pass Filter for each instrument using the specified laser.

For the Blue 488nm laser on the Aria, configured to 515/20, a GFP/YFP filter set is available.

Revised: March 14, 2016

The Flow Cytometry Core supports researchers at Penn State Health Milton S. Hershey Medical Center and Penn State College of Medicine, providing advanced instrumentation and expertise for flow cytometry analysis.

• Clinical Instruments:

  Three FACSCantos and three FACSLyrics are reserved exclusively for clinical use. Researchers may not use these instruments unless specifically authorized by Core personnel.

• Research Instruments:

  All other instruments, including the cell sorter, are designated for research purposes and must be scheduled in advance through iLab.

The facility is designed for user-operated workflows. Core personnel provide training to help researchers become proficient in experiment setup, sample acquisition, data analysis and archiving

Safety Guidelines

Safety is a top priority. The Core operates as a BSL-2 facility, and all users must follow established biosafety protocols, especially during cell sorting. Laser safety guidelines also apply. Please review the Safety Guidelines before using the facility.

Billing

Usage is billed monthly by Department and Principal Investigator. Billing is not itemized by technician or project; however, detailed reports can be provided upon request. All users must complete the required information in iLab for each session.

Data Management

Users are responsible for archiving all data. A 1 TB server is available for temporary data transfer from DIVA workstations. This space acts as a dropbox only and is not intended for long-term storage. Users may also transfer data to other portable storage media if desired. DIVA workstations retain data for up to 3 months.

The Flow Cytometry Core 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 ensure 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 core staff.

General Safety

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, PhD, Chair) before they are allowed to be used in the Flow Cytometry Core. Additional information can be found on the safety section of the Infonet (internal access only; login required).

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. 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+ Standard Operating Procedures will be required (these procedures are located in the 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 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 Clarity LIMS. Samples that have been fixed for analyzing or sorting on the Cell Sorter may be monitored and recorded to ensure that the fixation is adequate.

9. Work surfaces are to be decontaminated with 10 percent household bleach (FACS Clean) after any spillage of sample or hazardous material and after each user completes their 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 core in double-sealed containers per College of Medicine safety policy.

Laser Safety

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:

1. Access to the laboratory will be limited to only those people 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.

2. In such cases, the appropriate protective goggles or glasses will be made available and must be worn by those in the work area.

3. Only qualified personnel shall operate or adjust any laser.

Any questions regarding these guidelines may be directed to the Flow Cytometry Core manager, Nate Sheaffer, at nas2@psu.edu or 717-531-6908, or to the director of the Flow Cytometry Core, Todd Schell, PhD, at tschell@pennstatehealth.psu.edu or 717-531-8169.

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.

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 non-zero.

The three emergency steps are:

1. Immediately initiate first aid at the work site.

   1. Contaminated skin should be meticulously cleaned for 10 minutes using a povidone iodine solution (such as Betadine) and copious amounts of water.

   2. Contaminated eyes and mucous membranes should be irrigated for five minutes using water.

2. Notify your supervisor, if that person is immediately available. More importantly, go on to step 3.

3. Report to the hospital emergency department to activate the invasive accident protocol (needle-stick protocol), which will include evaluation, counseling and provision of antiretroviral treatment if deemed appropriate. Do this immediately (i.e., within the half-hour).

FluoroFinder

The FluoroFinder system allows researchers with experiment design using the College of Medicine’s flow cytometers.FluoroFinder offers training videos:

• An Introduction to FluoroFinder

• The Basics of Panel Design with FluoroFinder

Use Penn State’s FluoroFinder

Fluorochrome Specifications

BD Biosciences, makers of the flow cytometry equipment used at Penn State College of Medicine, has created a guide to its fluorochromes.

Search for “BD Biosciences Fluorochrome Specifications Chart” here

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 0.5 ml. Cell concentrations should be 0.5 to 1.0 X 106 cells per ml.

Fluorochrome Selection

Investigators should review the capabilities of the instruments before selecting fluorochromes and dyes to ensure that the instruments can excite and detect the dyes of choice. For multicolor experiments, it is imperative to consult fluorochrome guidelines or personnel in the facility for best possible outcomes.

Compensation Controls

Investigators must 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 being used. If samples are stained with more than one fluorochrome per test tube, investigators must bring in a sample stained with each fluorochrome individually. This is for compensation purposes.

If there are not enough positive stained cells for each fluorochrome, investigators may use antibody capture beads.

To make conclusions about the experiment, the proper controls are critical.

With questions or to learn what the appropriate controls are, call facility personnel at 717-531-6908.

Cell Surface Marker/Antigen Staining

For each control and test sample, prepare a single cell suspension of 1X106 cells in cold PBS. Wash cells once in at least 1 ml of PBS with 2 percent FBS/media, centrifuge cells and aspirate supernatant.

Resuspend cells in a final volume of 50μl containing the primary antibody at the correct titer and PBS with 2 percent FBS/media. Reference manufacturer’s guidelines but in general, incubate on ice or at room temperature for 20 to 30 minutes.

Wash cells twice in PBS with 2 percent 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 50μl containing the secondary antibody and PBS with 2 percent FBS/media.

Resuspend cells at a final concentration of 1 to 2 x 106 cells/ml in at least 0.4ml fresh 1 percent to 2 percent formaldehyde/PBS. Provide the proper controls and analyze by flow cytometry.

Cell Cycle

Two options are 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)

See protocols on intracellular staining and other methods here (UCLA).

Apoptosis Techniques

There are good commercial kits available. The core recommends using kits with Annexin V PE and 7AAD as the best fluorochrome combination.

Cells should be concentrated to at least 5 million cells/ml. Investigators who want to sort lots of cells in a short amount of time may concentrate cells even more (but the more concentrated cells are, the more likely they might form clumps). Investigators should remember to calculate how many total cells will be needed to analyze in order to sort the required number of target cells after the sort experiment. In general, lymphocyte-sized cells can be run at rates close to 30K per second, and cell lines can be run around 12K per second.

Cell media must be brought in the tubes or trays into which the cells will be sorted. Tubes should be polypropylene to avoid static charges and can vary in size. Media should fill about one-third of the tube space before collection begins.

It is required that cells are filtered before sorting. Clogs will be a problem to any sorting experiment and increase biohazard risks. 35 or 40μm filter cap tubes are available from BD Biosciences for this purpose.

Also, investigators must read and adhere to the safety guidelines before sorting unfixed cells, and the cell sorting experiment form must be completed before any sorting experiment is scheduled. This form must be submitted for review one week prior to scheduling the sorting experiment.

If, after the core’s review of the cell sorting experiment form, it is determined that the experiment is at the BSL2+ level, the investigator will be required to read and adhere to the Flow Cytometry BLS2+ Standard Operating Procedures for cell sorting, which is available in the cell sorter room.

Any request to sort live, primary human cells in the Flow Cytometry Core will require a review of the samples in question by the core director based on the information on the cell sorting experiment form and possible consultation with the principal investigator 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 this 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, investigators must contact the clinical trials coordinator for details on the testing process. Once tests are completed, a copy of the test report without patient ID will be signed by the requesting principal investigator and forwarded to the Flow Cytometry Core before the cell sorting experiment is performed.

The following boilerplate language can be used in grant applications when referencing the College of Medicine Flow Cytometry Core.

The Flow Cytometry Core Facility routinely analyzes both clinical and research samples in a clinically (CAP)–accredited facility. Equipment available for use by investigators includes: two three-laser 12-color BD FACSLyrics ; two three-laser, 10-color Becton Dickinson FACSCantos; one two-laser, six-color Becton Dickinson FACSCanto; one four-laser, 16-color Becton-Dickinson LSR Fortessa; and one five-laser, 23-color Becton Dickinson FACSymphony analyzer, a BD Rhapsody HT Single-Cell Analysis System for single cell capture and cDNA synthesis.

In addition, a four-laser, 16-color Becton Dickinson Aria III high-speed four-way sorter currently housed outside a biocontainment hood for BSL-1 sorts only, a three laser, 9-color FACS Melody sorter, and a BD FACSDiscover S8 Spectral sorter with Cellview Technology are available for operator-assisted live cell sorting.

Computer workstations equipped with multiple flow cytometry analysis software packages are available for data analysis.

More information about the core is available at https://med.psu.edu/research/core-facilities/flow-cytometry-and-cell-sorting.

Methods

• Isolation of intact RNA from murine CD4+ T cells after intracellular cytokine staining and fluorescence-activated cell sorting Shajo Kunnath-Velayudhan, Steven A. Porcelli. In Journal of Immunological Methods, Volume 456, 2018, pages 77-80, ISSN 0022-1759.

Introductions to Flow Cytometry

• Video Introduction to Flow Cytometry (ThermoFisher Scientific)

• Other Flow Cytometry Tutorials (ThermoFisher Scientific)

• Practical Flow Cytometry (free online book by Howard Shapiro)

Fluorochrome Choices and Controls

• See details on the fluorochrome choices with each piece of equipment under Instrumentation and Services

• Penn State FluoroFinder

• BD Biosciences resources (maker of equipment used at Penn State College of Medicine)

Cytoplasmic Staining Resources

• BD FACSelect Buffer Compatibility Resource

• PhosphoSitePlus

Safety

• The Laser Institute

• OSHA

• NIH Occupational Health

• CDC Lab Safety

Other Resources

• Current Protocols in Cytometry (see lab manager)

• Purdue University Cytometry Laboratories

• International Society for the Advancement of Cytometry

• Catalog of Free Flow Cytometry Software (Purdue University)

• College of Medicine Flow Cytometry Core on iLab

General Usage

To see available times for use of the equipment in the Flow Cytometry Core, use PSH credentials to access the calendar here. The calendar is view-only and is updated frequently.

Scheduling time for sample acquisition or data analysis is done by calling the core at 717-531-6908. Researchers are scheduled on a first-come, first-served basis. Scheduling is best done a few days in advance to ensure that sufficient time is available and that samples will be able to run within a reasonable time after they have been prepared.

For time-course studies, it is best to check with the facility well in advance to ensure that someone will be there on the days access to the cytometers is needed. 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.

Off-Hours Usage

The Flow Cytometry Core provides coverage during standard business hours through a specialist and a trained backup research associate for each instrument. However, on rare occasions, core staff may not be available even during regular hours. To avoid scheduling conflicts, researchers should consult the scheduling calendar well in advance to ensure their projects can be accommodated. Because of the nature of certain research projects, there are times when accommodations must be made for processing samples through the flow cytometers without the assistance of core personnel.

When core personnel are not present, only the Research FACSCanto II, LSR Fortessa, BD FACSymphony 17, BD FACSymphony 23, and BD FACSMelody are available for research use. Other instruments may be made available for clinical specimen processing under separate arrangements. The FACSDiscover S8 and the FACSAria Cell Sorter are never available for use without core personnel present.

Outside of normal business hours, such as evenings or weekends, only approved users may operate the flow cytometers independently after making prior arrangements with core personnel. All special arrangements must be scheduled in advance and approved by core staff. To qualify for off-hours access, investigators must complete a training checklist for the specific instrument they plan to use. Access to the facility is controlled by a keypad lock, and the code will be provided to these approved users. During these times, users are fully responsible for starting up and shutting down the instruments correctly. If any issues arise, the operator may contact the lab manager, Joe Bednarczyk, at 717-531-6908. If the problem cannot be resolved by phone, sample processing must stop until core personnel return.

Please check iLab or contact the core facility at 717-531-6908 or via email.

Note: “No-shows” and cancellations less than eight hours in advance may be charged for scheduled time.

To enter your flow cytometry usage time, please fill out the request form in iLab.