The first step of metastatic spread involves movement of cancer cells from the primary site into the bloodstream (intravasation) either directly or indirectly through the lymphatics (17 (link)). Measurement of MDA-MB-231/eGFP cells in the blood of tumor-bearing SCID mice was determined by three methods in two separate experiments. In one experiment (113007), untreated (n = 3) and 200 mmol/L bicarbonate–treated (n = 7) animals bearing primary tumors were euthanized after 36 d of tumor growth. At this time point, the primary tumors averaged 463 ± 33.5 mm3 in size in both groups. Blood was extracted by cardiac puncture into microfuge tubes and mixed with an equal volume of 100 mmol/L EDTA to prevent clotting. A blood volume of 10 μL was smeared on glass slides and dried. Green-fluorescing cells were counted manually under a fluorescent microscope at ×40 magnification. Nucleated cells from the remaining blood volume (∼ 300 μL) were obtained by centrifugation with Histopaque (Sigma), and resulting cells were resuspended in 96-well plates in 100 μL of PBS and measured on a Victor3 with excitation wavelength at 485 nm and emission at 535 nm. In another experiment (011508), blood was extracted by heart puncture from untreated and bicarbonate-treated mice (n = 8 each) by the same methods as above. Average tumor size was 121.8 ± 16.4 mm3. RBC were lysed with fluorescence-activated cell sorting lysing solution (BD Sciences) according to the manufacturer's instructions. Cells were counter-labeled with LDS-751 nucleic acid dye and analyzed by flow cytometry on a FACScan (BD Biosciences) with a 488-nm argon laser. LDS-751 emits at 670 nm upon excitation at 488 nm and is detectable with the fluorescence 3 detector. Nonspecific fluorescence was differentiated from the green fluorescent protein (GFP) signal by gating on cellular light scattering properties and LDS-751.