MPRI Flow Cytometry and Cell Sorting Facility

  • Location: 2102 Bioscience Research Building
  • Director: Ken Class, kclass@umd.edu
  • Office: 301-405-4593; Lab: 301-405-0398
  • 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 accomplished using Google Calendar.
  • FACSCanto II: 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.
  • FACSAria II: All FACSAria experiments must be arranged through the facility director.
You will be charged for time signed up unless canceled 24hrs in advance.

Rates

Instrument
Univ. of MD.
External Users
**Minimum Billable Time
FACSCAnto II
$20/hr
$50/hr
0.25 hr
FACSAria II
$40/hr
$50/hr
1.00 hr
FACSAria II sort setup
n/a
n/a
add 0.5 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 II will be able to do so after undergoing one-on-one training and demonstrating efficiency and instrument operation.

Sample Preparation

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

FACSAria II Operating Characteristics

Excitation sources available and commonly used fluorochromes (contact Ken Class regarding any additional dyes that are not mentioned in the table below):
Excitation Type
Wavelength
Commonly Used Fluorochromes
Coherent Sapphire
solid state
488nm
(blue)
FITC, GFP, Alexa Fluor 488, PE, PE-Texas Red, DSRedPerCP, PerCP-Cy5, 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)
-430, Cascade Blue, Pacific Blue, Dye Cycle Violet
Additional specifications:
Nozzle tip size
Approximate max Flow Rate and operating pressure
Sample Concentration (cells/ml)*
70mm
22,000 cells/second, 70 PSI
20-40x10^6
85mm
15,000 cells/second, 35 PSI
10-20x10^6
100mm
7,500 cells/second, 20 PSI
5-10x10^6
130mm
3,000 cells/second, 10 PSI
1-5x10^6
*For sorting experiments, it is preferable to prep cells toward the higher and 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, 0.5-5.0x10^6 cells/ml are the optimal concentration regardless of the nozzle tip size.

Contact Us

Ken Class
Director
kclass@umd.edu
301.405.4593
2102 Bioscience Research Building