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Flow Cytometry Facility

"ALL samples brought to our facility should be filtered with 70-micron cell strainer"

Overview of Services

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 real-time analysis of Ca2+ mobilization kinetics. The sorters, analyzers, and workstations allow for affordable, reliable, and accurate characterization of a wide variety of biological samples.

The services provided by the facility can be tailored to suit experimental designs in a variety of fields. We provide consultation on current protocols, analysis techniques, and data presentation. This resource plays a critical role in assisting SKCC members performing various research projects directly related to the understanding of the biology and treatment of cancer. Furthermore, the facility supports projects from investigators in Biochemistry, Genetics, Immunology, Medicine, Microbiology, Molecular Biology, Neurology, Pathology, Pharmacology, Surgery, and many other scientific fields. The facility currently has 5-laser-16-color BD AriaII high-speed sorter, 5-laser-16-color BD Fortessa, 4-laser-14-color BD LSRII, 3-laser-12 color BD Celesta and 3-laser-8-color BD Melody sorter.

Leadership

Jianke Zhang, PhD
Director
jianke.zhang@jefferson.edu

Staff

Lei Yu, PhD
Manager
215.503.3216
lei.yu@jefferson.edu

 

Amir Yarmahmoodi, BSc
Technician
215.503.4556
amir.yarmahmoodi@jefferson.edu 

Hours of Operation and Location

Hours Location

Monday - Friday

9:00 AM-5:00PM                        

233 S 10th Street, Room 606
Philadelphia, PA 19107

 

Links and Resources

 

FluoroFinder for Jefferson Researchers

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

BD Horizon Guided Panel Design

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

 

Useful protocols and flow handbook from R&D system

 

       

 

Extra filters besides the standard configurations are available for the LSRII, Fortessa and Aria II sorter

LP BP
450 440/40
495 510/20
505 515/30
525 525/50
550 575/26
595 585/42
600 605/40
610 610/20
630 660/20
635 660/40
690 670/30
750 700/50
  780/60

 

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. You can contact your local friendly SKCC flow cytometry staff members to assist you in experimental design. We strongly suggest you use the Fluorofinder websita listed above to design your panel.

 

How do I access the facility?

The facility is located at 233 S. 10th St. Philadelphia, PA 19107 in the Bluemle Life Science Building Room 606. The analyzers in the facility are available 24 hrs a day, 7 days a week. 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 check our institutional biosafety committee policies before coming to the facility: Jefferson Biosafety. There are additional flow cytometry related biosafety guidelines to consider: Flow Cytometry Biosafety.

 

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

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

 

 What instruments does the facility have:

Our facility has three analyzers and one sorter. We also have built a specific website to help you pick the right fluorophores that fit the equipment. Only proper fluorophores will generate a good result.

https://app.fluorofinder.com/jefferson/panels/new

Besides the flow cytometry machines, we also have two workstations with FlowJo to analyze flow data

 

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 oncompensation 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, 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 the higher purity, the machine will discard any droplet that contaminated with negative target 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. Furthermore, the Sidney Kimmel Cancer Center Flow Cytometry facility is now certified with a >90% success rate of placing single cells onto the Advalytix  AmpliGrid PCR slide.  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.

PrimeFlow is a new technology that can measure gene expression and surface markers simultaneously. 

 

Contacts

Name Role Phone Email Location
Lei Yu, Ph. D.
Manager
 
215.503.3216
 
lei.yu@jefferson.edu
 
BLSB 606A