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The FACSAriaIII is a digital, fixed-alignment cell sorter equipped with a Violet (405nm, 50mW), Blue (488nm, 20mW), Yellow-Green (561nm, 100mW) and Red (633nm, 18mW) laser, capable of measuring up to 17 fluorescent parameters in addition to FSC and SSC. The Aria is housed in a Class I safety cabinet.

The sorter offers up to 4-way sorting into a variety of tubes or deposition of single-cells into a variety of receptacles using the Automated Cell Deposition Unit e.g. tissue culture plates, 96-well PCR plates and microscope slides, including multi-well chamber slides.

Independent temperature control for both the sample and collection tubes, plus sample agitation, helps to maintain the viability of your sample and improve the success of your sort.

facsariaiii.jpg

The fluorochromes listed in the table below are examples of reagents which can be measured in each detector, but does not represent an exhaustive list. Please ask if you are unsure whether a particular reagent can be detected or not.

If you wish to measure any fluorescent parameters which are not listed above, please contact the Facility Manager as we may have additional optical filters to fit your requirements. A more detailed instrument configuration including the dichroic mirrors and bandpass filters can be found below.

Some general information is provided below to assist you in planning your sorts, however, please come and discuss your experiments well in advance, especially before applying for a new type of sort.

About the machine

  • The AriaIII is capable of sorting up to 4 populations at the same time into 0.2ml, 0.5ml, 1.5ml tubes or 5ml FACS tubes, or up to 2 populations into 15ml Falcon collection tubes.
  • The AriaIII can also sort into a variety of plates and slides; please discuss this with Helen before planning a sort to make sure your chosen receptacle is suitable.
  • As a rule of thumb, it takes ~2-3min to put 1x106 cells through the sorter when sorting at 70psi and ~4-5min when sorting at 20psi.
  • The AriaIII has four nozzle sizes: 70µm, 85µm, 100µand 130µm.
  • When sorting in ‘4-way purity’ mode with a 70µm nozzle at 70psi e.g. for lymphocytes, you can expect to sort up to ~3x106 cells/5ml FACS collection tube.
  • When sorting in ‘4-way purity’ mode with a 100µm nozzle at 20psi e.g. for DCs, you can expect to sort up to ~1x106 cells/5ml FACS collection tube.
  • Calcium- and magnesium-free Dulbecco’s PBS is used for the sheath fluid – it is the user’s responsibility to check that this buffer is appropriate for their cell type.
  • A long clean will be performed prior to the first sort of each day – this is a thorough clean of the system’s fluidics, although it does not prepare the sorter for a truly aseptic sort. If your sort is the second sort that day, and would like an additional long-clean to be performed, that is possible, although please note that you will be charged for the time this takes (~45min º £75).

Sample preparation

  • As the Sort Yield (i.e. how many cells are sorted in total) is influenced by many things including threshold rate, flow rate, sample quality, target population frequency etc., it is prudent, especially for a primary experiment, to assume that the Sort Yield will be ~50% of the Theoretical Yield.
  • The more you can enrich your sample for the population of interest, the quicker (& therefore cheaper) the sort will be - your cells will be happier too!
  • All samples must be filtered prior to acquisition. It may be necessary to filter your samples again during the sort, so please bring additional filters with you. Suitable filters include:
    • BD Falcon 35µm filter cap/PS FACS tube (VWR, 21008-948)
    • BD Filcon cup, available in a variety of mash sizes, including 10µm, 20µm, 30µm and ≥50µm.
    • Partec CellTrics, available in a variety of mash sizes, including 10µm, 20µm, 30µm and ≥50µm.
  • Samples must be diluted in a media which does not contain phenol red e.g. calcium- and magnesium-free PBS or HBSS supplemented with FCS; it is recommended that you also include 10-20mM HEPES.
  • If your sample contains particularly sticky cells e.g. cells which have previously been frozen, please include 1-2mM EDTA in all steps of the sample preparation. DNase can also be used if EDTA proves to be ineffective for your sample type.
  • In the first instance, please dilute your sample at 15-20x106/ml for sorting at 70psi & 5-10x106/ml for sorting at 20psi. Please bring additional media in case it is necessary to dilute your samples further.
  • You should always include a live/dead marker in your staining panel (who wants to sort dead cells!) this is particularly important when you are analysing rare cell populations (≤1%) since mAb-fluorochrome conjugates can bind non-specifically to dead cells bind, leading to staining artefacts.
  • You must provide appropriate controls for your experiment for both compensation and setting the gates e.g. single stained cells or beads and FMOs- if you are unsure what controls to bring, please ask.

 Collection tubes

  • Cells adhere less to Polypropylene (PP) tubes than Polystyrene (PS), therefore it is recommended that you use PP tubes for both your sample and collection tubes – this is particularly important when small numbers of cells are involved.
  • If you expect to sort <300,000 cells with the 70 µm nozzle or <200,000 with a 100µm nozzle, it is recommended that you sort into 1.5ml tubes, to maximise your post-sort recovery.
  • To prevent your sorted cells sticking to the walls of the collection tubes, they need to be coated with FCS or media. As a general rule, it takes a volume of 10-15% of the collection tube to “wet” the tube.
  • If appropriate, collection tubes should be provided with sufficient FCS so that your sorted sample will have a sufficient concentration FCS at the end of the sort to keep your cells happy.  

Feedback regarding your post-sort cell count, viability or sterility would be much appreciated.

FACSAriaIII configuration

Laser PMT Dichroic mirror Bandpass filter Range, nm

Violet

A

B

C

D

E*

F

750LP

735LP

635LP

600LP

502LP

-

780/60

710/50

655/30

610/25

525/50

450/40

750-810

685-735

640-670

600-623

502-550

430-470

Blue

A

B

655LP

502LP

-

695/40

530/30

488/10

675-715

515-545

Yellow-Green

A

B

C

D

E

735LP

685LP

630LP

610LP

-

780/60

710/50

670/30

616/23

585/42

750-810

685-735

655-685

610-628

564-606

Red

A

B

C

755LP

690LP

-

785/62

730/45

660/20

755-816

708-753

650-670