FACS Detection of RASSLs in Blood Cells

By Charles H. Redfern & Bruce R. Conklin

February 28, 1999

Note: This protocol has been used successfully to detect Ro1 intetO-Ro1/MMTV-tTA mice. The original data were cut from of our 1999Nature Biotechnology paper (Redfern et al.) at the request of our reviewers.In this section of the paper we demonstrated inducible Ro1 expression inlymphocytes of MMTV-tTA/tetO-Ro1 mice by flow cytometry. The meanpercentage of cells with Ro1 expression in total peripheral blood lymphocyteswas 10.8 ± 3.7% (SD, n = 13) and was almost equally divided betweenT- and B-lymphocytes. This expression was completely suppressed by feedingthe mice doxycycline.

FACS remains a powerful means of detecting RASSLs in rare subpopulationsof cells, such as stem cells, certain immune cells and neural cells. Ifyou have any corrections or additions to this protocol, please e-mail usso that we can assist other people trying to use RASSLs in rare subpopulationsof cells.

Methods (short version)

Flow cytometry. Mouse whole blood (200 mlas mixed in PBS containing heparin (2 units/ml) at 4°C, centrifuged,and lysed in standard lysis buffer (150 mM NH₄Cl, 10 mM NaHCO₃,and 0.4% EDTA). The white blood cells were resuspended in Dulbecco''''s modifiedeagles medium (2% fetal calf serum), centrifuged three times, stained withphycoerythrin-conjugated M1-Ab (Custom conjugation by Chromaprobe MountainView CA, or Molecular Probes, Oregon), and preserved in PBS with 1.0% paraformaldehydeat 4°C until analyzed by flow cytometry (Becton Dickinson).

Methods (long version)

1. Cut mouse tail with sharp razor.
2. Collect 200 ml of blood in 15 ml Falcon conical tube containing 12 ml of fresh PBS and heparin 1-2 units/ml.
3. Invert tube several times, store on ice while cutting other tails.
4. Spin Falcon tubes in centrifuge at 1000-1200 rpm for 5 minutes.
5. Pour off supernatant (careful, the pellet is not tight) and resuspend in 3 ml of 1x Lysis Buffer.
6. Let stand in Lysis Buffer for 5-10 minutes at room temperature.
7. Spin for 3 minutes at 1000 rpm.
8. Resuspend in media (DME + 2% FCS) and spin again for 3 minutes at 1000 rpm.
9. Resuspend in Fluorescent Staining solution (~1 ml) and shake on ICE for 20-30 minutes (in Light-tight Box).
10. Pellet, wash in 2 ml of media, pellet, and wash in 1% PFA/PBS.
11. Pellet, and resuspend in 500 ml of 1% PFA/PBS in FALCON TUBE (2054).
12. Cover with tin foil and refrigerate until FACS time.

Lysis Buffer (10x)

• Add to 1 liter millipore water
• Store at 4°C.
• Dilute in millipore water

Antibody Staining Solution

• Mix 1 ml of media solution with approximately 1 mg of antibody for each reaction.
• Keep fluorescent antibody light protected at all times.

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