Cell separators reserve

The service will allocate the requested hours according to the availability of existing equipment and the waiting list of users.

  • The request form is not only necessary for the reservation of cell separators, but will also collect essential information for the cytometry service to design the most appropriate separation strategy.
  • Hours of equipment use and service provision will be billed according to current rates.
  • Bookings should be cancelled, whenever possible, within 24 hours.
  • The preparation of the sample will be the responsibility of the user, who must bring them already prepared and ready for analysis and/or separation. In the technologies section you will find some advice and indications for the adequate preparation of the samples.
  • It is very important to provide the cytometry service with the appropriate controls, as well as a test sample, so that the service can make the calibrations and the experiment template, and can test the proposed separation strategy.
  • The images resulting from the separation work can be exported as FCS files so that the user can have them available and analyze them in the computers enabled for this purpose.

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Requirements for any cell separation

Negative control: unlabeled cells or cells labeled with the control isotype. (In case of GFP parental cells).

Compensation controls, if necessary: If the staining is with several fluorochromes, monolabeling of each fluorochrome will be necessary.

The cell death marker will be added by the cytometry service and its choice will depend on the combination of fluorochromes in the sample.

Controls only need a final volume of 500µl with 500,000 cells. They can be brought in FACS tubes in FACS Buffer or Sorting Buffer.

Bring the cells at 4°C and protected from light.

  • Samples in sterile tubes on ice.
  • Always filter them through cellstrainer.
  • The concentration will be 30x106 cells/ml. When the cells are larger than 20 microns the concentration will be about 10x106 cells/ml.

In 15 ml culture tubes put 1 ml of culture medium containing 20% serum or sterile FBS.

You can calculate the approximate volume of cells to be collected and use smaller tubes (FACS tubes, eppendorf...), if desired consult with the cytometry service.

  • Cytometer tubes: Ref. 352052 BD Falcon
  • 15ml polypropylene tube: Ref 430791 Corning or similar
  • Cell strainer 50 microns (50 u): Ref. 352350 BD Falcon
  • EDTA 0.5M pH8.0 (4X100ml): Ref. 15575-020 GIBCO
  • Cytometer tube with cell-strainer cap: Ref. 352235 BD Falcon

1x PBS (no Ca/Mg++)-1mM EDTA-25mM HEPES pH 7-0.5% Fetal Bovine Serum (Heat Inactivated)+ 1% antibiotics (penicillin and streptomycin).

  50 ml

1x PBS (Ca/Mg++ free)

Up to 50  mL

2.5mM EDTA (stock 500mM)

250 uL

25mM HEPES pH 7.0 (stock 1M)

1.25

0.5% FBS (Inactivated)

250 uL

1% antibiotics (pen and strep)

500 uL

Store at 4°C.

If the cells are adherent increase the EDTA concentration to 5 mM.

If there are a large number of dead cells it is recommended to add 10U/mL DNAase II.

Nozzle orifice size

Cell type

Final concentration (per mL)*

70um

Lymphocytes, thymocytes

8-15 x 106

80um

Activated cells, small cell lines

7-10 x 106

100um

Adherent cells, large cells

5-9 x 106

 

Nozzle and pressure selection

The FACSAria II cell separator has three different nozzle orifice sizes 70, 85 and 100 microns, and can separate (sort) cells using different pressures.

The size of the nozzle and the pressure to be used are directly related to the size of the cells to be separated. The larger the cell size, the larger the nozzle orifice, the lower the pressure to be used and therefore the lower the separation rate.

There are general rules to determine the appropriate nozzle size and pressure, however, variations can be made depending on the resistance or fragility of the cells to be separated. Therefore, it is very important to inform the cytometry department if problems are experienced after separation.


70 microns, high pressure separation (70psi)

  • Primary tissue cells: thymus, spleen, bone marrow.
  • Peripheral blood cells.
  • Cell lines derived from B and T cells.


85 microns, medium pressure separation (45psi).

  • Those listed above but have been transfected/transduced with expression vectors.
  • Cells from primary cultures.
  • Activated cells.


100 microns, low pressure separation (20psi)

  • Most cell lines.
  • Adherent cell lines.
  • Large and delicate cells of primary tissues (neurons, dendritic cells).
  • Newly transfected cells expressing GFP.


Cell
concentration

  • High pressure: 8-15 million/ml (max. 22,000 cells/sec).
  • Medium pressure: 7-10 million/ml (max. 12,500 cells/sec).
  • Low pressure: 5-9 million/ml (max. 6,500 cells/sec).

* These values correspond to the final concentration after filtering of the sample by the cytometry service, so the sample should be provided at twice the concentration.