Flow Cytometry and Human Immune Monitoring Shared Resource

ALL samples brought to the flow facility should be filtered with 70-micron cell strainer

CLICK HERE to report any problems encountered during instrument use.

PUBLICATION ACKNOWLEDGEMENT: "This project utilized the Flow Cytometry and Human Immune Monitoring Shared Resources at the Sidney Kimmel Cancer Center, supported by the NCI grant 5P30CA056036-17."

 

Flow cytometry

The Sidney Kimmel Cancer Center facility provides state-of-the-art fluorescence-activated cell sorting and analysis capabilities to investigators of the Sidney Kimmel Cancer Center and the Philadelphia region. The most commonly used applications are:

  • multi-color cell surface phenotyping
  • detection of intracellular cytokines and signaling molecules
  • transfection measurements
  • studies of apoptosis, cell cycle analysis, cell proliferation, and Ca2+ mobilization kinetics

 

The sorters, analyzers, and workstations allow for affordable, reliable, and accurate characterization of a wide variety of biological samples.

We provide a wide range of consultation services including:

  • experimental design
  • antibody panel creation
  • current protocols
  • analysis techniques and data presentation

 

The services provided by the facility can be tailored to suit research projects in a variety of fields including; Biochemistry, Genetics, Immunology, Medicine, Microbiology, Molecular Biology, Neurology, Pathology, Pharmacology, Surgery.  This resource facilitates study design, data capture, and presentation to SKCC members in order to better understand the biology and treatment of cancer resulting in high-impact publications and funded grants.

 

The facility currently contains a wide variety of instruments:

Cell Sorters

Cell Analyzers

Other Instruments

  • Nanosight 300 small particle analyzer
  • autoMACS pro cell separator

 

Human immune monitoring

The Sidney Kimmel Cancer Center facility provides cutting-edge immune monitoring services to clinical and basic science  investigators throughout the greater Philadelphia region.  Our most common assays include:

  • patient sample processing and short-term storage (blood, urine, stool, etc)
  • phenotypic and functional flow cytometry (T cell, monocytes, and dendritic cells)
  • multiplex luminex and single-plex ELISA
  • GeoMX digital spatial profiling

The core works closely with investigators to design research projects, optimize experimental protocols, and interpret data.  Our main users include:

  • monitor patients enrolled in clinical trials testing novel therapeutics
  • prognostic and therapeutic response biomarker discovery
  • deeper understanding of interactions between the host immune response and tumor that occur in the tumor microenvironment, lymphatic tissue, and peripheral blood  

 

Study specific, pilot-projects, and advanced immune assays can be developed as needed.  

The core is positioned to coordinate data collection by multidisciplinary cores, i.e. Translational Pathology and MetaOmics. 

This facility is dedicated to meeting the current and future needs of both researchers and clinicians as they generate complex immunological hypotheses required to drive cutting-edge science facilitating informed clinical decisions.  

 

Leadership

Larry Harshyne, PhD; Co-Director - larry.harshyne@jefferson.edu

Brian Montoya, PhD; Lab Manager - brian.montoya@jefferson.edu

 

Staff

Brenda Vidals, MS; Flow Cytometry Lab Coordinator - brenda.vidals@jefferson.edu

Margaret Lippincott, MS; Human Immune Monitoring Lab Coordinator - margaret.lippincott@jefferson.edu 

 

Hours of Operation

Monday-Friday, 9am-5pm

Location

Bluemle Life Sciences Building, 233 S 10th Street, Philadelphia, PA 19107

Flow facility - room 606

Immune-monitoring facility - room 822

 

Links and Resources

 

           the website has all our individual machine settings that can help you design your panels based on the available lasers and filters.

           this website will help you design a multicolor panel considering resolution impact.

 

If the current machine configurations can't accommodate your experiment design, extra filters besides the standard configurations are available for the LSRII, Fortessa, and Aria II sorter.

 

 

Frequently Asked Questions:

How should I design my flow cytometry experiment for success?

First, you'll need to decide which parameters you are interested in researching. Then you'll need to find that molecule attached to a fluorescent probe for that particular parameter. We strongly suggest that you contact SKCC flow cytometry staff members to assist you in experimental design. 

 

How do I access the facility?

The facilities are located at 233 S. 10th St. Philadelphia, PA 19107 in the Bluemle Life Science Building. The analyzers in room 606 are available 24 hrs a day, 7 days a week and coordinators may drop off patient samples off in room 822 after hours.  However, you must obtain a 5-digit security code to access the facility outside of normal work hours.

 

Are there any biosafety concerns?

Yes, there are many things to consider before bringing your samples to the facility. Please consult with our flow cytometry staff and biosafety prior to using this facility: Jefferson BiosafetyFlow Cytometry Biosafety.

 

What biosafety paperwork do I need to fill out before I can flow?

You must fill out a flow cytometry biosafety form before your experiment and give a copy to the Sidney Kimmel Cancer Center flow cytometry staff.

 

 

What do I need for cell sorting?

Your sample can be in 15ml, 5ml tube, or 1.5-2 ml Eppendorf tube. You'll need a negative control. The negative control will be unstained cells, not your experimental negative. You'll also need a positive control for each fluorochrome you are considering. These are called compensation controls. For example, if you want to set up an experiment and use 5 fluorochromes you'll need 6 controls to properly set up the cytometer (1 negative and 5 compensation controls). For information on compensation, you can look here. If compensation doesn't make sense you can always stop by the facility and talk to the flow operator. When you think you have an understanding of compensation try this Quiz. Additionally, your sample should be sterile and the cells should be resuspended in either PBS or media with no more than 2% serum. You'll also need a collection tube with a few milliliters of media. The collection tube can be an Eppendorf tube, a 5ml tube, or a 15ml tube. The cell sorter has an additional function that allows it to sort into 6, 24, 48, and 96 well plates as well as custom slides.

If you only doing one population sorting, we still suggest you use 5ml tube so we can put the collection tube far away from the waste stream to prevent any spread contamination in case of nozzle clog. You should also increase the serum concentration in your collection tube because the carryover sheath flow will dilute the culture medium. Also, a higher concentration of protein can help to release the charge that sorted cells may carry.

Before you bring the sample to our facility, you have to filter your cells to remove any big particles that will clog the nozzle. We suggest 70 micron cell strainer.

 

How many cells will I get from cell sorting?

You'll need to do a bit of math to get the answer. If you have 10 million cells and 50% are positive for your marker of interest then you'll receive 5 million sorted cells. The other 5 million cells are collected in the waste container. If you don't know how many cells you have and you don't know the percentage of cells you're interested in, then you don't know how many cells you'll get from sorting.

The actual number will vary depends on many facts: The purity requirement. To achieve higher purity, the machine will discard any droplet that contaminated with negative targets along with your positive target; The quality of your sample. If there is lots of debris, the sorter may sort only at low efficiency. If the cells are fragile, some of them cannot survive the high-stress condition when they pass through the sort. The machine will automatically generate a sort report showing the number of cells that have been sorted. We have seen the recovery rate varied within a big range.

 

How long will it take me to sort?

It also depends. If the sample is free of debris and cells are not fragile, we can sort and over 10000 events/sec without negative effects on the purity. There are 11-grade speeds we can choose from.

If the nozzle gets clogged during the sort, it obviously will take some time to clean (disassemble the nozzle, sonication, and re-alignment). If you book a time for sorting, we will overbook a time slot for you at the beginning, the final charge will be based on the actual machine time that has been used.

 

How many populations can I sort?

You can simultaneously sort up to 4 separate populations.

 

How many parameters can I quantify?

Currently, the high-speed cell sorter can simultaneously measure 16 separate fluorochromes plus Forward and Side scatter. That makes 8 separate parameters you can measure. The analyzers can accommodate up to 16 fluorochromes and 18 parameters.

 

What new technologies exist in flow cytometry?

  • The Aria II cell sorter allows for single-cell sorting into 96 well plates for cloning purposes. 
  • The Fortessa has a high-throughput platform that allows analysis of samples in a 96-well plate.
  • Techniques are available to place single cells onto the Advalytix  AmpliGrid PCR slide with >90% success rate.  The AmpliGrid slides are used in subsequent PCR or RT-PCR reactions on the AmpliSpeed slide cycler.  We recommend AmpliGrid for single-cell molecular analysis because you can visually verify correct cell placement on the slide and the analysis is more sensitive in the 1 ul reaction volume.
  • RNA detection using PrimeFlow or RNA Scope. 

 

Contacts

Name Role Phone Email Location
Larry Harshyne, PhD
Co-Director
 
215-503-9893
 
larry.harshyne@jefferson.edu
 
M-41 JAH
 
Brenda Vidals, MS
Lab Coordinator
 
215-503-4556
 
brenda.vidals@jefferson.edu
 
606 BLSB
 
Margaret Lippincott, MS
Human Immune Monitoring Research Assistant
 
215-955-4262
 
margaret.lippincott@jefferson.edu
 
822 BLSB
 
Brian Montoya, PhD
Sr Research Investigator
 

 
brian.montoya@jefferson.edu
 
606 BLSB