Antibody Staining

 

Necessary controls:

  • Cells only
  • 1° Ab only
  • 2° Ab only
  1. If adherent cells, trypsinize, scrape, or treat with EDTA to get single cell suspension.

  2. Count cells, at least 2 x 106 cells will be needed for each sample.

  3. Spin cells down (5 min @ 100 x g in tabletop centrifuge).

  4. Resuspend cells to 2 x 106 cells/200 μl in 2% FBS/PBS (FACS buffer).

  5. Transfer 200-μl volumes to individual 1.5 mL microcentrifuge tubes.

  6. Add 10 µl 1° Ab at the desired dilution to appropriate tubes. Incubate 1 hr at 4°C.
    Optimal concentration for each antibody will need to be determined by the end user.

  7. Add 1 ml FACS buffer and spin down. (3 min @ 800 x g in Eppendorf microcentrifuge)

  8. Aspirate the supernatant.

  9. Wash with 1 ml FACS buffer, spin down again.

  10. Aspirate the supernatant.

  11. Add 2° Ab at desired concentration in 100 μl of FACS buffer + 1% BSA to appropriate tubes.

  12. Incubate at 4°C for 30 minutes.

  13. Repeat steps 7-10.

  14. Resuspend in 500 μl FACS buffer.

  15. Transfer into 12 x 75 mm polystyrene FALCON # 2024 snap-cap, 5 ml test tubes. If fixing cells before analysis, do not add propidium iodide.

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