Cell Sorting
Cell Sorting
Lighthouse operates four high speed cell sorters: a 16-color FACS Aria Fusion and a 17-color FACS Aria III, both from Becton Dickinson, as well as a MoFlo Astrios EQ and a 15-color CytoFlex SRT benchtop sorter, both from Beckman Coulter. Due to their complexity, these machines are normally operated by members of the Core Facility. The CytoFlex does offer possibilities of self-operation to specially-trained users.
Note: Because the sorters are heavily booked, it is best to reserve times on the instruments as early as possible.
If you need more information as to which combinations of dyes or fluorescent proteins it is possible to use, please contact us. The best time to do this is when first planning a new experiment.
Available Cell Sorter
Lasers / Configurations:
Cell Sorters: FACS Aria Fusion MoFlo Astrios CytoFlex SRT |
Cell Sorter: FACS AriaIII
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Examples of Possible Dyes/Proteins
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Near UV: 375 nm* *colinear with 405 nm laser |
DAPI, Hoechst (including detection of SP cells and cell cycle analysis) |
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405 nm
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405 nm
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DAPI, Alexa 405, CFP, Brilliant Violet Dyes, Pacific Blue, Live/Dead Aqua, Zombie Aqua, etc. |
488 nm | 488 nm |
FITC, Alexa 488, Brilliant Blue 515, GFP, YFP, CFSE, PerCP, PerCP-CY5.5 |
561 nm | 561 nm |
PE, PE-Texas Red, PE-CY5, PE-CY5.5, PE-CY7, dsRed, tdTomato, mCherry, etc. |
638 nm | 633 nm |
APC, Alexa 633/647, TO-PRO3, APC-CY7, APC-H7, Alexa 680/700, etc. |
For additional help with panel design, the configurations of all Lighthouse sorters and analysers are now listed on the Fluorofinder website. To access information about the cell sorters please follow the links below:
MoFlo Astrios EQ
FACS Aria III
FACS Aria Fusion
CytoFlex SRT
Other possibilities for the cell sorters are:
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Cell/well cloning directly onto slides or into i.e. 96- or 384-well tissue culture plates (up to 1536 Wells on the MoFlo Astrios)
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4-way/6-way sorting: Up to 4 populations can be sorted simultaneously on the Arias and the CytoFlex, and up to 6 populations on the MoFlo Astrios.
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Index Sorting - the ability to look up the phenotype of a cell after cloning/sorting into a well plate.
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Small particle detector - MoFlo Astrios EQ
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Biosafety Hood - FACS Aria Fusion and CytoFlex SRT
For more information about setting up your samples for sorting and the best buffers to use, with your samples please also see:
Sample Preparation
Sample tubes
Because of their differing electrostatic properties, it is highly recommended that you use polystyrene tubes (clear) as sample tubes, and polypropylene tubes (opaque) for sample collection, Sample tubes should be 12 x 75 mm in size, while collection tubes can be either 12 x 75 mm in size, 15 ml conical tubes for large samples, or 1.5 ml tubes for small samples.
Sorting into Plates
When sorting into plates for subsequent live cell culture, be sure to prepare the wells beforehand so that they are ~75% full. This makes it easier to sort into the well, with less chance of the droplet landing on the side of the well. For special plates or formats it is helpful to bring by an empty plate to set up the stage positions ahead of time.
How to Select the Correct Nozzle Size
As a general rule of thumb: The cell size should not exceed one-fifth of the nozzle diameter. Using the wrong nozzle size causes fanning of the side streams, poor sort purity and nozzle clogging.
Nozzle Size
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70 µm
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85 µm (Arias) |
100 µm
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130 µm (Arias) |
Pressure (pounds per sq. in.) | 60-70 psi | 45 psi | 20-25 psi | 10 psi |
Max. Cell Diameter |
14 µm | 17 µm | 20 µm | 26 µm |
Max. Events/Sec. | 22.000 - 30.000 | 11.000 | 7.000-10.000 | 3.000 |
Max. Events/Hour | 7-9x107 | 3-4x107 | 2x107 | 1x107 |
Starting Cell Conc. of Sample | 3-5x107/mL | 1-2x107/mL | 5-7x106/mL | 2-4x106/mL |
Approx. Size of Drop | 1 nL | 2 nL | 4 nL | 11 nL |
Cell Concentration Post-Sort | 1x106/mL | 0,5x106/mL | 2,5x105/mL | 1.1x105/mL |
If you are unsure which nozzle size you will require, just ask one of the sort operators and they will be happy to advise you. They can also help you to determine what diameter your cells have while in suspension.
Biosafety
The cell sorters normally operate at pressures of 60 - 70 psi, and can aerosolize a portion of the sample. The Lighthouse cell sorting service is classified to a Biosafety Level of 2. Therefore only cells of risk groups 1 and 2 can be sorted. .
It is necessary to determine the risk potential of the cells prior to sorting to take appropriate precautions. For this reason, please fill out the following form:
Lighthouse Biosafety Form
Sort experiments of any biosafety level can only be carried out after filling out the proper form.
Note:
It is not necessary to fill out the checklist for every appointment. However, it should be filled out for each project, including experiments involving different cell types, treatment or sample origin.
FACS Analysis
For information about our FACS Analysers please see the
FACS Analysis page.
Offline Analysis
The Core Facility also has offline analysis workstations with the flow cytometry software programs FlowJo, FCS Express, Diva, Sony Analysis Software and Kaluza available for general use.