Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures.

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G(0) state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.

Characterization of magnetite nanoparticles for SQUID-relaxometry and magnetic needle biopsy.

Magnetite nanoparticles (Chemicell SiMAG-TCL) were characterized by SQUID-relaxometry, susceptometry, and TEM. The magnetization detected by SQUID-relaxometry was 0.33% of that detected by susceptometry, indicating that the sensitivity of SQUID-relaxometry could be significantly increased through improved control of nanoparticle size. The relaxometry data were analyzed by the moment superposition model (MSM) to determine the distribution of nanoparticle moments. Analysis of the binding of CD34-conjugated nanoparticles to U937 leukemia cells revealed 60,000 nanoparticles per cell, which were collected from whole blood using a prototype magnetic biopsy needle, with a capture efficiency of >65% from a 750 µl sample volume in 1 minute.

An automated, pressure-driven sampling device for harvesting from liquid cultures for genomic and biochemical analyses.

Here we describe an automated, pressure-driven, sampling device for harvesting 10 to 30 ml samples, in replicate, with intervals as short as 10 s. Correlation between biological replicate time courses measured by microarrays was extremely high. The sampler enables sampling at intervals within the range of many important biological processes.

Enhanced leukemia cell detection using a novel magnetic needle and nanoparticles.

Acute leukemia is a hematopoietic malignancy for which the accurate measurement of minimal residual disease is critical to determining prognosis and treatment. Although bone marrow aspiration and light microscopy remain the current standard of care for detecting residual disease, these approaches cannot reliably discriminate less than 5% lymphoblast cells. To improve the detection of leukemia cells in the marrow, we developed a novel apparatus that utilizes antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPION) and directed against the acute leukemia antigen CD34, coupled with a "magnetic needle" biopsy. Leukemia cell lines expressing high or minimal CD34 were incubated with anti-CD34-conjugated SPIONs. Three separate approaches including microscopy, superconducting quantum interference device magnetometry, and in vitro magnetic needle extraction were then used to assess cell sampling. We found that CD34-conjugated nanoparticles preferentially bind high CD34-expressing cell lines. Furthermore, the magnetic needle enabled identification of both cell line and patient leukemia cells diluted into normal blood at concentrations below those normally found in remission marrow samples. Finally, the magnetic needle enhanced the percentage of lymphoblasts detectable by light microscopy by 10-fold in samples of fresh bone marrow aspirate approximating minimal residual disease. These data suggest that bone marrow biopsy using antigen-targeted magnetic nanoparticles and a magnetic needle for the evaluation of minimal residual disease in CD34-positive acute leukemias can significantly enhance sensitivity compared with the current standard of care.