Brian Crucian | PubMed Health

PubMed2006

Rapid flow cytometry method for quantitation of LFA-1-adhesive T cells.

Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an "adhesive" state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-microm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3+ CD4+ or CD8+ CD19- CD16- CD45RO+ CD62L+ CD27+ CD57-. A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.

Cell Adhesion↗

PubMed2005

Reduced gravity evaluation of potential spaceflight-compatible flow cytometer technology.

BACKGROUND: The presence of a spaceflight-compatible flow cytometer onboard the International Space Station would be extremely beneficial for in-flight medicine and reduced gravity research. In the past, commercially available flow cytometers were not suitable for use during spaceflight due to their size, weight, power, and sheath fluid requirements. Recent advances in cytometer technology have enabled features that now make a spaceflight-compatible cytometer possible. We evaluated a small, robust novel cytometer with design features that minimize or eliminate many incompatibilities with spaceflight. This cytometer is highly miniaturized and lightweight, does not require sheath fluid, and uses a low-energy diode laser. The ability to achieve laminar particle flow without sheath fluid is important because an instrument using large liquid volumes and producing an equivalent amount of hazardous liquid waste would not meet spaceflight constraints. METHODS: For this study the cytometer was modified so that stained liquid cell samples could be delivered during reduced gravity. The cytometer was then evaluated onboard the NASA KC-135 reduced gravity research aircraft. The KC-135 uses a parabolic flight path to generate essentially zero gravity conditions for 30-s increments. Typically 40 parabolas are flown per mission, resulting in approximately 20 min of reduced gravity during which research may be performed. During this evaluation, bead-based cytometer precision, photomultiplier tube linearity, and leukocyte immunophenotype analysis were performed during reduced gravity. The flight data were then compared with ground-based control data and data generated using a reference cytometer (Beckman-Coulter XL). RESULTS: This novel cytometer functioned well during reduced gravity and produced data comparable to those of ground-based controls with only minor caveats. The reduced gravity cell immunophenotype data were indistinguishable from ground control data and reference cytometric data. Bead-based instrument precision (coefficient of variation) was slightly increased during reduced gravity operation, but not to a degree that would affect most common flow cytometric applications. The ability of the instrument to collect absolute cell counts was validated. CONCLUSIONS: This study represents the first generation of real-time flow cytometry data during zero gravity. With modifications, the evaluated cytometer technology could be the basis from which an operational spaceflight-compatible flow cytometer is developed.

Adult↗