Flow Cytometry Resources

Applications

Applications range from cell cycle analysis, apoptosis, fluorescent protein expression levels, immunophenotyping, etc… to sorting up to 4 different viable cell sub-populations based on expression patterns using fluorescent dyes or proteins.

Specifications 

The Core Facility’s FACS Aria II is a cell sorter allowing analysis and sorting of samples with up to 7 colors/ 9 parameters into tubes or multi-well plates.

The blue laser (488 nm) module provides up to 5 detectors and filter sets for a number of common fluorophores (FITC, GFP, Alexa Fluor 488, PE, PE-Texas Red, DSRedPerCP, PerCP-Cy5, PE-Cy7, PI, 7AAD). Filter sets include (in order of detection) :
LP735 + 780/60, LP655 +695/40, LP610 + 616/23, LP556 + 585/42, LP502 + 530/30.

The red laser (633 nm) module provide 2 additional detectors and filter sets (Common fluorophores are APC, APC-Cy7, Cy5, Alexa Fluor 647). Filter sets include (in order of detection) :
LP735 + 780/60, 660/20.

4 Nozzle sizes allow for a range of cell types and sizes to optimize cell sorting and survival for culture:

• 70 mm : max flow rate of 22,000 cells/second at 70 PSI of operating pressure. Sample concentration should be between 20-40x106 cells/ml.
• 85 mm : max flow rate of 15,000 cells/second at 45 PSI of operating pressure. Sample concentration should be between 10-20x106 cells/ml.
• 100 mm : max flow rate of 7,500 cells/second at 20 PSI of operating pressure. Sample concentration should be between 5-10x106 cells/ml.
• 130 mm : max flow rate of 3,000 cells/second at 10 PSI of operating pressure. Sample concentration should be between 20-40x106 cells/ml.

Sample Preparation

Samples for the FACSAria must be pre-filtered through ≤70 µm cell strainers prior to being loaded onto the instrument. BD Falcon tubes with filters (cat#352235) or 40 µm cell strainers (Cat# 352340) are proper options to filter your samples.

Samples should come in 12x75 flow cytometry tubes to avoid unnecessary pipetting and biohazardous transfers before loading samples into the sorter.

Samples for sorting are generally prepared in PBS+1% FBS unless otherwise necessary.

For sorted cells, collection tubes (12x75) should contain about 1ml of media complemented with serum and antibiotics as in your usual culture to help maintain cell viability and sterility.

If you are planing on performing analysis only, samples can be fixed in 2-4 % paraformaldehyde and run the following day, if it’s more practical.

Proper controls are necessary for a correct sort or cell analysis. Proper sample controls for setup and compensation are as follow :

• Unstained cells only control
• Isotype controls
• Compensation controls of single color stained cells for each dye in your experiment. Beads stained with the same dyes as your cells are also acceptable but are not suitable for cells expressing fluorescent proteins as those have different emission spectra from similar commercial dyes (GFP is actually different from FITC…). This is necessary for multi-color experiments to subtract (compensate) the non-specific overlapping part of the dye’s emission spectra.

User Information

For more information on the Aria II system :
https://www.bdbiosciences.com/documents/BD_FACSAria_II_cell_sorter_brochure.pdf
https://www.bdbiosciences.com/documents/BD_FACSAria_II_User_Guide.pdf

Applications

The BD FACS Celesta is cell analyzer designed to make multicolor flow cytometry more accessible, with the ability to detect and analyze up to 14 parameters in a single sample. Our department’s Celesta is equipped with 3 lasers, Blue (488 nm), Violet (405 nm) and Red (640 nm) and up to 12 colors. This system is only partially software driven and requires some level of hands-on steps and controls compared to a FACS Canto, for example, but is simple to use. The Celesta is also equipped with BD’s High Throughput Sampler (HTS) for automated sample loading from multi-well plates and allows for increased sample acquisition speed and efficiency.

Specifications

The Core Facility’s FACS Celesta is a cell analyzer allowing analysis and sorting of samples with up to 12 colors/ 14 parameters from tubes or multi-well plates using BD’s HTS. 

Configuration

The Blue Laser (488 nm) module provides up to 5 detectors and filter sets for a number of common fluorophores (FITC, GFP, Alexa Fluor 488, BB515, PE, PE-Texas Red, DSRedPerCP, PerCP-Cy5, PE-Cy7, PI, 7AAD, etc…). The Blue laser module is also responsible for detection of Side Scatter (SSC). Filter sets include (in order of detection) :
LP750 + 780/60, LP670 +695/40, LP550 + 575/25, LP505 + 530/30. 

The Violet Laser (405 nm) module provides up to 5 detectors and filter sets (common fluorophores include BV786, BV650, BV605, BV510, V500, BV421, V450, Pacific Blue, etc…). Filter sets include (in order of detection) :
LP750 + 780/60, LP655 +670/30, LP595 + 610/20, LP505 + 525/50, 450/40.

The red laser (640 nm) module provides 3 additional detectors and filter sets (Common fluorophores are APC-Cy7, APC-H7, APC-R700, Alex Fluor 700, APC, Alexa Fluor 647, etc…). Filter sets include (in order of detection) :
LP750 + 780/60, LP690 + 730/45, 670/30.

Software 

The FACS Celesta is controlled by BD’s FACSDiva software for acquisition and analysis.

User Information

For more information on the BD FACS Celesta system, visit:
https://www.bdbiosciences.com/en-us/products/instruments/flow-cytometers/research-cell-analyzers/facscelesta

For information on the FACSDiva software, visit:
https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software#Overview