• About the Core
• User Guide
• Fees, Policies and Reservations
• Sort Information and Biohazard Form

Location: 1300 York Ave (at East 69th Street) Sixth Floor, Lab A-610C

Hours: Cell sorting 10-5 Monday through Friday (by appointment)

Lab Manager: Steven Merlin (212) 746-3387 [email protected]

Lab Director: Mikhail Roshal MD, PhD (212) 746-2096 [email protected]

About the facility:

The facility is equipped with a FACS Aria II and is operated by a dedicated operator with extensive expertise in cell sorting. The sorter is available to WCMC researchers on the fee for service basis. Consultations on optimal sorting approaches are available. More extensive collaborative projects requiring substantial input from the core director in regards experiment design for optimal population detection are also welcome.

Prior to signing up for the sort all users must complete a sorting information form Instrumentation:

BD FACSAria II-SORP

It has five excitation lines at 355nm (UV), 405nm (Violet), 488nm (Blue), 561nm (Green-Yellow), and 640nm (Red). Click on the following link to view current detection filter layout. We have several additional filters to facilitate detection of fluorescent proteins. It can collect up to 14 fluorescent parameters and sort 1-4 separate populations simultaneously, or perform single cell or mixed population sorting into 6,12,24,48,96 and 384 well plates as well as microscope slides.

Sample Preparation and Sorting:

Cells: Samples should be filtered prior to sorting through filter top tubes (Falcon 12X75mm Cat#2235). These sterile tubes have a 35-micron mesh filter in the cap.

Cell Concentration: Your sort results will be improved if cells are appropriately concentrated. For small cells that can run with either a 70 or 85 micron nozzle approx. 1-2X10^7 cells per ml resuspended in HEPES buffer or similar low protein buffered media is suggested (see recipe below). For larger or more fragile cells (larger then human monocyte) requiring 100 or 130-micron nozzle 3-5x10^6 cells per/ml is recommended. Bring extra sterile buffer for dilution if needed. No phenol red pH indicator should be used as it increases background fluorescence. Minimum sample volume is 500ul. For single cell sorting into microtiter well plates, concentration should be1-5 x106 cells per ml. For primary cells and cell lines that have a propensity to form cell aggregatesthere are a number of reagents that can be employed to reduce or eliminate theformation of aggregates. Pluronic F-68 sold by Sigma (Cat #P5556, 100ml sterile 10% solution) is a non-ionic, non-toxic surfactant ideal for cell sorting andsample acquisition. Dilute into the sample to give a final concentration of 1%vol/vol. Other reagents are: DNAse, EDTA (1 mM) or PBS w/o Ca++ or Mg++containing 0.1% BSA or FCS. Other commercially available products are Accutase for confluent cell lines and Accumax for Adherent cell lines. Both products are available through Innovative Cell Technologies/Phoenix Flow Systems http://www.innovativecelltech.com/

Sorting on FACS Aria II:

Sample Tubes: Falcon polystyrene tubes (12X75mm, Cat#2054 or 2058, sterile with cap) or Falcon15mL conical, sterile (Cat#2096). Collection Tubes: Same as above except for 4-way sorting tubes must be 12X75mm or 1 mL Bio-RadFor recovery of cells for further culture collection tubes should contain an appropriate volume of HEPES buffered media supplemented with 20% FCS or another buffered media appropriate for your downstream application. To keep cells "happy" post sort at least 1ml of media per 10^5 cells sorted is recommended. If purity determination is desired after the sort no phenol red pH indicator should be used.

Rate: The current rate for cell sorting is available on the WCMC Department of Pathology & Laboratory Medicine Flow Cytometry website under "Cell Sorter Core Facility Fees and Policies Data, Acquisition Template and Instrument Settings storage

Data may be analyzed by FACS DiVa or FlowJo or any other software capable of handling FCS 3.0 format. If your lab needs a copy of FlowJo, please contact the facility manager for more information on how to obtain. Data files can be transferred via LAN to your lab server in fcs 3.0 (flow cytometry standard) format. Due to high risks from the proliferation of computer viruses infecting flash memory drives, this method is currently not available as a method of data file transfer. Large data files can be burned to a CD-R, CD-RW or DVD.

HEPES BUFFER:

1XPBS (No Ca or Mg), 1mM EDTA, 25 mM HEPES pH 7.0 (Invitrogen 15630-106), 1% FCS (heat inactivated)
Filter through 0.2-micron filter