Contact

UWCCC Flow Cytometry Core Facility

NIH / NIAID / DAIT platforms

Issue 3, Spring 2013

Follow us on Facebook

Call us at

(608) 263-0313

Instrumentation 18 parameter Cell Sorter

BDFACS Aria II

15 parameter benchtop flow cytometer

BD LSR II

6 parameter benchtop flow cytometer

BD FACS Calibur

High-content kinetic Imaging System

BD Pathway

Cell counter Nexcelomm

10 parameter benchtop flow cytometer

MACS Quant10

We are located at 7003 WIMR (office) 7016 WIMR (lab) 1111 Highland Ave Madison, WI 53706

Fluorescent microscope

Olympus

FlowJo Site License The University of Wisconsin has acquired an institutional, shared-cost software license. Register any computer on- or off-campus for a $240 annual fee. Turn in a dongle for a credit for 4 years of use. The license ensures immediate access to any new versions of the software on any Mac, PC or Linux system.

We have opened business on the new ARIA III cell sorter. This sorter is placed in a Level II biosafety cabinet providing sterile conditions for sorts and BSL2 containment for human samples. All human samples and virally transfected cells are sorted on this instrument.

Visit us at

www.uwhealth.org/flowlab

see page 2

The UWCCC Flow Lab and the UW Biosafety Office

have updated institutional guidelines for live cell sorting and flow cytometry to reflect recent changes in NIH/ISAC recommendations. See page 3 for details.

15 parameter Cell Sorter in BSC

BDFACS Aria III

http://immport.niaid.nih.gov

see page 2

Flow Newsletter Issue 3 / Fall 2013

UWCCC Flow 2

Flow Cytometry Data Analysis Tools

Fluorish is an online flow cytometry network and and panel design tool. It provides reagent information and access to core instrument configurations.

NIH/NIAID has constructed a website where users can import and deposit their raw flow files. The site offers other analysis tools as well for both flow cytometry (FLOCK) and genetic analysis (ie. MHC Analysis, tagSNP). There is a tentative plan that all journals will require deposition of raw flow files so that other investigators can perform secondary analyses after publication.

http://immport.niaid.nih.gov

SPICE Simplistic Presentation of Incredibly Complex Evaluations

SPICE is a data mining software to analyze and interpret highly complex multi-color flow cytometry data. It can process large polychromatic FlowJo files and organizes the normalized data graphically. It simplifies analysis of highly sophisticated assays and may aid discovery of potential new correlations. http://exon.niaid.nih.gov/spice

Flow Cytometry Clustering without K • computationally determines the number of

unique populations in high dimensional flow data using a rapid binning approach

• maps populations across independent samples

• calculates summary statistics • find the most informative parameters • reduces subjective factors in manual gating • can handle non-spherical hyper shapes

Flow Newsletter Issue 3 / Fall 2013

UWCCC Flow 3 UWCCC Flow

Biosafety Guidelines for Live Cell Sorting Institutional protocols updated reflecting recent changes in NIH/ISAAC guidelines

What has not changed: • all live cell sorting experiments have to

be included in the biological safety protocol and pre-approved by the IBC prior to appointment requests

• this is a longstanding institutional regulation and is now strictly enforced to ensure safety of both core users and staff at our high-traffic BSL2 facility

What has changed: Instrument assignment / containment: • all live cells of human origin must be sorted

in a biological safety cabinet (BSC) • most BSL-2 samples will be sorted in the

BSC including any virally transfected samples

Pre-screening requirements: • all primary human samples including

embryonic stem cell lines must be screened for infectious diseases to prevent aerosolization of BSL-3 agents (ie. HIV-1,2, HepB, HepC, HTLV-1,2)

• all virally transfected samples and infected material must be pre-tested to minimize risk of transmission

Improved communication: • researcher is responsible for providing

detailed information on any live samples delivered to the facility to enable proper risk-assessment and to ensure assignment of appropriate staff and containment equipment

Why Biosafety is so important in live cell sorting? Live cell sorting involves the purposeful aerosolization of cell suspensions. The instrument generates a stream of single cell droplets in order to deflect single particles. The single-droplet stream typically contains ‘satellite’ droplets, which fall in the size experimentally proven to be the most successful in aerosol transmission of infectious particles to the lower airways. It is also not uncommon for aerosol incidents to occur during regular sorter operation as a result of clogging, air introduction and stream instability. Live cell sorting therefore is a serious health risk to the operator. The availability of novel reagents to achieve efficient genetic engineering have greatly increased the number of sort samples associated with agents considered BSL2+ when in aerosol (lentiviral systems, CMV, EBV, HTLV, etc.). Stem cell technologies have introduced an increasing number of samples expressing oncogens and tumor-suppressor knock-down sequences. Human clinical trials in general are sampling a population typically at increased risk of contracting or originally carrying infectious diseases (patients affected by cancer, immunosuppression, etc.). For all these reasons, biosafety is one of the most important components for live cell sorting.

Aerosol produced after deflection from waste catcher. Cytometry Part A 52A:122-130 (2003)

Flow Newsletter Issue 3 / Fall 2013

UWCCC Flow 4

Ask the Flow Core Staff

Have a question about flow cytometry, imaging or about our other core services? Contact us!

Manish Patankar, PhD patankar@wisc.edu Faculty Supervisor

Dagna Sheerar, BS dsheerar@wisc.edu Education, Consultation, Administration, Billing, Communication, Outreach, BD Pathway, FlowJo site license, Multi-color Assay Development

Karen Ersland, PhD ke@wisc.edu Cell Sorting, Daily Quality Control, Assay Services, Hands-on Training, Data Analysis, ModFit, Figure preparations

Faye Bruggink, BS fbruggink@wisc.edu Online schedule, Assay Services, MACSQuant Protocol Development

Erika Heninger, PhD eheninger@wisc.edu Cell Sorting, Biosafety Protocol Amendments, Consultation, FlowJo Site License, Data Analysis, Figure preparations, Multi-color assay development

“Introduction to Flow Cytometry” Lecture Series Spring/Summer schedule May 20, 22, 24 June 24, 26, 28 July 22, 24, 26

Flow Core NewsLetter, Issue 3/ Spring 2013 Editor: Erika Hėninger, eheninger@wisc.edu

Data Analysis & Consultation Need help with data analysis or guidance to navigate the myriad of new options in flow cytometry & imaging? Schedule a consultation and we will be happy to assist you with both experimental design and interpretation of data. We also offer assistance with preparation of publication-ready figures and other flow cytometry data presentations.

Core Services

Assay Services

Our facility offers standardized assay services including everything from sample processing to data interpretation. Besides the assured Quality Control and high-throughput options, this service has been providing great value to laboratories without equipment or trained staff to perform flow cytometry & imaging. Contact us to see what we can offer to develop customized protocols for your experimental models.

Training & Education

For new users we offer a monthly lecture series titled “Introduction into Flow Cytometry”. We provide one-on-one, hands-on training sessions on all of the instruments. Assisted services are also available for inexperienced users and experienced users with unfamiliar assays, on schedule or upon request. Training sessions are customized to our users’ individual experimental needs.

Dagna Sheerar Facility Manager Dagna has been doing flow cytometry since

1999. She had managed a flow laboratory in Canada for 3 years before returning to the UWCCC Flow Lab. She was promoted to facility manager in April 2011. Dagna loves building multi-color flow cytometry panels and is interested in highly complex assays. Her door is always open to discuss new projects and new collaborations.

Flow Publications from campus Publish your panel! Multi-­‐color  panel  for  human  Treg  cells Nettenstrom  L,  Alderson  K,  Raschke  EE,  Evans  MD,  Sondel  PM,  Olek  S,  Seroogy  CM.  An  optimized   multi-­‐parameter   flow   cytometry   protocol   for   human   T   regulatory   cell  analysis   on   fresh   and   viably   frozen   cells,   correlation   with   epigenetic   analysis,   and  comparison  of  cord  and  adult  blood.  J  Immunol  Methods.  2013  Jan  31;387(1-­‐2):81-­‐8.    Th1  TH2  flow  panel  for  human  T  cells                                    Zuleger  CL,  Albertini  MR.  OMIP-­‐008:  measurement  of  Th1  and  Th2  cytokine  polyfunctionality  of  human  T  cells.  Cytometry  A.  2012  Jun;81(6):450-­‐2.      Minimal  amounts  of  antibody  Sharma  D,  Eichelberg  MR,  Haag  JD,  Meilahn  AL,  Muelbl  MJ,  Schell  K,  Smits  BM,  Gould  MN.  Effective  flow  cytometric  phenotyping  of  cells  using  minimal  amounts  of  antibody.  Biotechniques.  2012  Jul;53(1):57-­‐60.