The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Surface & Cytoplasmic Staining

The following protocol is for simultaneous surface and cytoplasmic staining (for CD3) (formaldehyde/NP40 method).

Reagents

  • Monoclonal antibodies conjugated to FITC, PE, Cy5, etc.
  • Staining/wash medium: 1X Hanks 1% BSA 0.1% sodium azide pH 7.36
  • 3.7% formaldehyde in PBS (Sigma Chemical)
  • 0.01% NP 40 (Sigma Chemical)

Staining

  1. Add monoclonal antibodies for surface antigens (PE and Cy5 conjugated) to aliquots of 5 x 105 cells.
  2. Wash once with staining/wash medium.
  3. Add 100µl of 3.7% formaldehyde to cell pellet.
  4. Incubate at room temperature for 5 minutes.
  5. Wash twice with staining/wash medium.
  6. Add 100µl of 0.01% NP 40 to cell pellet.
  7. Incubate at room temperature for 5 minutes.
  8. Wash twice with staining/wash medium.
  9. Add monoclonal antibody for intracellular staining (FITC conjugated).
  10. Incubate at room temperature for 25 minutes.
  11. Wash twice with staining/wash medium.
  12. Add 200µl of staining/wash medium.
  13. Analyze on flow cytometer within 3 days.

References

A Poggi et al. In Vitro Expansion of CD3/TCR- Human Thymocyte Populations That Selectively Lack CD3g Gene Expression: A Phenotypic and Functional Analysis. J. Exp. Med. 172:1409-1418, 1990