Fixation and Permeabilization

1. Intraprep permeabilization reagent (Beckman Coulter, Miami, FL).

2. Phosphate-buffered saline (flow PBS): prepare with 5.6 g sodium phosphate dibasic anhydrous, 35.48 g sodium chloride, 2.8 g bovine albumin, and 4 g sodium azide. Add water to 4 L and adjust to pH 7.4 with HCl.

3. Fluorochrome-labeled surface markers: CD3-FITC, CD19-PerCP, and CD34-APC (BD Bioscience, San Jose, CA).

4. Fluorochrome-labeled isotype controls: IgG-FITC, IgG-PerCP, and IgG-APC (BD Bioscience).

5. Primary monoclonal mouse antibody: CRKL (32H4) (Cell Signaling Technology, Beverly, MA).

6. Primary polyclonal rabbit antibodies: phospho CRKL (Tyr 207) (Cell Signaling Technology).

7. Secondary antibodies: goat anti-rabbit conjugated to phycoerythrin (PE) fluoro-chrome (Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti-mouse conjugated to PE fluorochrome (Jackson ImmunoResearch Laboratories, West Grove, PA).

8. Sorvall cell washer 2 (Thermo Electron Corporation, Asheville, NC).

9. Imatinib mesylate (Gleevec) (Novartis, East Hanover, NJ).

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