MPRI Flow Cytometry and Cell Sorting Facility

 

  • Location: BRB 2102
  • Director: Ken Class, kclass@umd.edu
  • Office: 54593, Lab: 50398
  • General laboratory operating hours: 10-6 Mon-Fri (extended hours available on request).

 

Please complete pre-experiment information here and submit to kclass@umd.edu.

 

All signups are implemented using Google Calendar. You will receive an invite to the FACSCanto Google calendar once a valid gmail account is provided. You must continue to confirm dates/times with the facility director until 24/7 access to the facility has been granted. All FACSAria experiments must be arranged through the facility director.

 

You will be charged for time signed up for unless canceled 24hrs in advance.

MPRI Flow Cytometry Facility User Rates eff. Feb 2014

Instrument Univ. Of MD. External **Min Billable Time
FACSCanto II $20/hr $50/hr 0.25 hr
FACSAria II $40/hr $50/hr 1.00 hr
FACSAria II sort setup     add 0.50 hr
Luminex Magpix $12/plate $24/plate n/a

 

** Minimum Charge of $50 for External users on FACSaria and FACSCanto

All chargebacks are calculated on a 0.25hr basis with the exception of the Luminex instrument. Samples will be run and data collected by the facility Director, Ken Class. Frequent regular users who wish to have 24/7 access to the FACSCanto will be able to do so after undergoing one-on-one training and demonstrating proficiency in instrument operation.

Sample Preparation

All samples to be run on the FACSAria must be pre-filtered through no greater than 70um strainers prior to being run on the instrument. (BD Falcon tubes, cat# 352235 are a preferred source. The flow lab has samples of these to try, but it will be mandatory to eventually purchase your own).

  • Samples to be delivered in 12x75 tubes. (Polystyrene only for FACSCanto II)
  • See page 3 regarding optimal cell concentrations.
  • Required samples (controls/setup) Controls/compensation tubes. All experiments must include:
    • Cells only control
    • Isotype controls
    • Compensation controls, single stained tubes utilizing each dye in your experiment. This is mandatory in multi-color (2 or more dyes) experiments since emission spectra from dyes overlap and non-specific fluorescence must be subtracted (compensated) from the various photodetectors.
  • Sort samples are generally prepared in PBS+1%FBS. Please contact Ken if alternative media is preferred/needed.
  • Sort collection tubes (12x75) should contain approx 1ml of media + 20% serum + antibiotics (pen-strep, gentamycin), as this provides a cushion for the sorted cells and helps maintain sterility. Analysis-only samples not requiring viability can be fixed in 2% paraformaldehyde and run the following day, if necessary.

Examples of Potential Flow Cytometric Applications

Up to 9-color measurement of surface and/or internal antigens on FACSAria (8-color on FACSCanto)

  • Cell cycle analysis (fixed or live cells)
  • Measurement of fluorescent protein expression
  • Apoptosis and cell death measurement
  • Fluorescence Resonance Energy Transfer (FRET)
  • Ion mobilization measurements (i.e. Calcium flux)
  • Isolation of sub-populations of interest based on fluorescent characteristics

(Cell Sorting) into tubes or tissue culture plates.

Data analysis programs available: FACSDiva software, Flowjo (coming soon), Microsoft Office products.

FACSAria Operating Characteristics:

Excitation sources available and commonly used fluorochromes:

Coherent® Sapphire™ solid state 488nm (blue) FITC, GFP, Alexa Fluor-488, PE, PE-Texas-Red, DSRedPerCP, PerCP-Cy5.5, PE-Cy7, PI, 7AAD
JDS Uniphase™HeNe air cooled  633nm (red) APC, APC-Cy7, Cy5, Alexa Fluor 647, Topro-3
Point Source Violet solid state 407nm (violet) Alexa Fluor- 405 and
-430, Cascade Blue, Pacific Blue, Dye Cycle Violet
Contact Ken at kclass@umd.edu regarding any additional dyes that are not mentioned in the table above.
Nozzle tip size Approx. max Flow Rate and operating pressure (cells/second) Sample Concentration (cells/ml)*
70mm (70 PSI) 22,000 20-40x106
85mm (35 PSI) 15,000 10-20x106
100mm (20 PSI) 7,500 5-10x106
130mm (10 PSI) 3,000 1-5x106

** For sorting experiments, it is preferable to prep cells toward the higher end of the concentration range if possible. Extra media can be brought to the sorting facility for diluting the sample if necessary.

 

** For “analysis only” experiments, 0.5-5.0x106cells/ml are the optimal concentration regardless of nozzle tip size.