)

Requirements for any cell separation

  • Controls

    Negative control: unlabeled cells and cells labeled with isotype control (for GFP parental cells).

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

    The cell death label will be added by the Flow Cytometry Service and its choice will be depend on the combination of fluorochromes present in the sample.

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

    The cells should be stored at 4ºC and protected from light.

  • Samples to be separated

    Samples in sterile tubes in ice.

    They should always be filtered with a cell strainer.

    The concentration should be 30 x 106 cells/ml. When the cells are larger than 20 microns, the concentration should be around 10 x 106 cells/ml.

  • Collection tubes

    In 15-ml culture tubes, put 1 ml culture medium containing 20% serum or sterile FBS. You can calculate the approximate volume of cells you are going to collect and use smaller tubes (FACS, Eppendorf tubes); if you wish, check with the Flow Cytometry Service.

  • Material

    Cytometry tubes: Ref. 352052 BD Falcon

    15 ml polypropylene tube: Ref. 430791 Corning or similar

    50 micron (50 u) cell strainer: Ref. 352350 BD Falcon

    0.5M EDTA, pH 8.0 (4 x 100 ml): Ref. 15575-020 GIBCO

    Cytometry tube with cell strainer cap: Ref. 352235 BD Falcon

  • Basic Sorting Buffer

    1x PBS (Ca/Mg++ free)-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 4ºC

    If the cells are adherent, the concentration of EDTA should be increased to 5 mM.

    If there is a large number of dead cells, adding 10U/mL DNAase II is recommended.

  • Optimum concentration of cells

    Nozzle orifice size

    Cell type

    Final concentration (per mL)*

    70 um

    Lymphocytes, thymocytes

    8-15 x 106

    80 um

    Activated cells, small cell lines

    7-10 x 106

    100 um

    Adherent cells, large cells

    5-9 x 106

     

Contactar

Goretti Azparren
Secretaria
Citometría de flujo

General contact:
Avenida Pío XII, 55
31008 Pamplona
España

+34 948 194 700 Ext. 81 1009
mgazparren@unav.es

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