The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Immunofluorescence Staining

The following protocol is the courtesy of Dr. Elizabeth Field, University of Iowa Department of Internal Medicine.

  1. Add 0.5 x 106 - 1 x 106 cells to 12 x 75 mm polystyrene tubes.
  2. Spin down cells at 300 x g for 10 minutes at 4°C.
  3. Remove supernatant.
  4. Resuspend cell pellet in 50µl of staining buffer (1X PBS / 1% FCS) containing conjugated monoclonal antibodies each at ≤1µg / 1 x 106 cells (or use dilution determined by titration for optimal staining). If using biotin conjugate, always stain with this antibody first and by itself.
  5. Incubate on ice 20-30 minutes.
  6. Wash once (twice if not underlaying with 100% FCS) by spinning down cells, removing supernatant and adding 1ml 1X PBS / 1% FCS + 0.5ml 100% FCS underlay or add 2ml 1X PBS / 1% FCS with no underlay.
  7. Spin down cells at 300 x g for 10 minutes at 4°C.
  8. If using biotin conjugate, resuspend cell pellet in 50µl of staining buffer (1X PBS / 1% FCS) containing streptavidin conjugated dye plus other directly conjugated antibodies, repeat steps 6 through 8.
  9. Resuspend cell pellet in 300µl of tissue culture media, Hank's Buffered saline, 1X PBS or formaldehyde fixative (analyze unfixed cells on the same day).
  10. An unstained sample or negative Ig control and single stained samples for each conjugated dye are also necessary for setting up the flow cytometer. (See Suggestions for Successfully Running Samples)

Note: Bovine Serum Albumin (BSA) may be substituted for Fetal Calf Serum (FSC). Propidium Iodide can be added to unfixed stained cells to differentiate live from dead cells.