September, 2020

Staining protocols for flow cytometry

 Staining for cell surface molecules

Molecules can be detected directly by fluorochrome labelled antibodies or indirectly using a fluorochrome labelled antibody to detect the primary antibody.

Step by Step Protocol:
  • Approximately 1x106 living cells are required for staining.
  • Cells are pipetted into a FACS tube (Falcon BD Biosciences) and centrifuged (1200 rpm/300xg, 5 min).
  • The supernatant is decanted and the cells shortly resuspended (1x106 cells in 50 l)
  • Add fluorochrome-conjugated antibody in titrated amounts or according to the recommendations of the supplier (1 g antibody/ 1x106 cells is commonly used)
  • Incubate for 10min at 4C in the dark (most antibodies stain perfectly at 4C, other conditions may apply, ask recommendations of the antibody supplier)
  • avoid intensive light to prevent photochemical inactivation of fluorochromes
  • Wash cells 2 times with 2 ml FACS buffer at 300xg, 5min, 4C
  • For indirect staining incubate with secondary antibodies
  • Resuspend cells in 400 μl FACS buffer for flow cytomety and keep cells on ice in the dark
  • Samples must be filtered through a cell strainer (30m) immediately before analysis to avoid clumping of the samples
FACS buffer:
  • Dulbecco's PBS (DPBS) without Mg2+ or Ca2+
  • 1% (w/v) heat-inactivated FCS
  • 0.09% (w/v) sodium azide
  • Adjust buffer pH to 7.4 - 7.6, filter (0.2 μm pore membrane), and store at 4C

 Detection of intracellular proteins
  • For intracellular antigen detection approximately 3 x 106 cells are required for staining
  • Cells are fixed using 0.01% (w/v) formaldehyde for 10-15 minutes (this will stabilise proteins), or alternatively fixed using "Cytofix" (BD Biosciences) for 10-15 min . All incubations are done at 4C.
  • For permeabilization cells are incubated with detergents (this will disrupt cell membrane).

    A, Triton or NP-40 (0.1 to 1% in PBS ). These will also partially dissolve the nuclear membrane and are therefore suitable for nuclear antigen staining. It should be noted that loss of cell membrane and cytoplasm will result in decreased light scattering and also in reduced non-specific fluorescence.
    B, Tween 20, Saponin, Digitonin and Leucoperm are mild membrane solubilisers. Use at 0.5% (w/v) in PBS. These give large enough pores for antibodies to go through without dissolving the plasma membrane. Suitable for antigens in the cytoplasm or the cytoplasmic surface of the plasma membrane. Also suitable for soluble nuclear antigens.
    C, Alternatively, "Cytoperm" (BD Biosciences) can be used as detergent.

    For permeabilization with detergents, cells are incubated for 20-30 min at 4C

  • Cells are washed 2-times with 2 ml "Fix/Perm wash buffer" (BD Biosciences)
  • Cells are incubated with fluorochrome-conjugated antibody for 30 min at 4C (or according the recommendations of the supplier). Direct staining is recommended, indirect staining may induce high background.
  • Cells are washed 2 times with 2ml of "Fix/Perm wash buffer"
  • Resuspend cells in 400 μl "FACS-Buffer" (BD Biosciences) and keep cells on ice in the dark
  • Samples must be filtered through a cell strainer (30 μm) immediately before analysis to avoid clumping of the samples.