Fluorescence-based Cell Research Articles

Species Tropism of Chimeric SHIV Clones Containing HIV-1 Subtype-A and Subtype-E Envelope Genes

To analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant SIV/HIV-1 (SHIV) clones were constructed from two HIV-1 subtype-A isolates (HIV-1SF170 and HIV-1Q23–17 from individuals in Africa) and two HIV-1 subtype-E isolates (HIV-19466 and HIV-1CAR402 from AIDS patients in Thailand and Africa), respectively. These four SHIV clones, designated SHIV-A-170, SHIV-A-Q23, SHIV-9466.33, and SHIV-E-CAR, contain envelope (env) genes from the subtype-A or -E viruses. Interestingly, SHIV-A-170, SHIV-A-Q23, and SHIV-9466.33 were restricted for replication in cultures of macaque lymphoid cells, whereas SHIV-E-CAR replicated efficiently in these cells. Additional studies to define the block to replication in macaque cells were focused on the subtype-E clone SHIV-9466.33. A SHIV intragenic env clone, containing sequence-encompassing V1/V2 regions of HIV-1CAR402 and V3/V4/V5 regions of SHIV-9466.33, infected and replicated in macaque lymphoid cells. These results indicated that the sequence-encompassing V1/V2 region of HIV-19466 was responsible for the block of the SHIV-9466.33 replication in macaque cells. Analysis of viral DNA in acutely infected macaque cells revealed that SHIV-9466.33 was blocked at a step at/or before viral DNA synthesis, presumably during the process of virion entry into cells. In a fluorescence-based cell–cell fusion assay, fusion pore formation readily took place in cocultures of cells expressing the SHIV-9466.33 env glycoprotein with macaque T-lymphoid cells. Taken together, these results demonstrated that the block of SHIV-9466.33 replication in macaque cells is at an early step after fusion pore formation but before reverse transcription.

In vitro cytotoxicity testing with fluorescence-based assays in cultured human lung and dermal cells.

An in vitro study using human cultured cells was conducted to determine the reliability of fluorescence-based cell viability indicators with traditional in vitro cytotoxicity testing methods. Human lung epithelial carcinoma (A549) cells, and human embryonic skin (WS1) and lung (HFLI) fibroblasts were studied in culture to evaluate their potential to screen for cytotoxicity and to compare to previous protocols conducted in our laboratory. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and fluorescent-labeled probes were used to assess toxicity. Eight chemicals, including mercuric chloride, copper sulfate, sodium fluoride, thioridazine HCl, paraquat, amitriptyline-HCl, verapamil-HCl and chloroquine sulfate, were tested with each cell line using calcein-AM and Sytox. The data suggest that fluorescent probes are sensitive indicators of cytotoxicity and contribute to understanding the mechanisms for each chemical. In combination with previously published reports, the similarity of results among cell lines may be explained by the origin of the cell lines rather than by the diversity of the methods and indicators employed.

Centrifugal assay for fluorescence-based cell adhesion adapted to the analysis of ex vivo cells and capable of determining relative binding strengths.

Cell adhesion assays are widely used to identify novel cellular ligands, novel cell surface receptors for these ligands and to elucidate the mechanisms responsible for the underlying cellular and molecular interactions. We report here the development of a novel centrifugal assay for fluorescence-based cell adhesion (CAFCA) that offers a number of advantages over the currently available assays. CAFCA is based on two centrifugation steps: one to allow for the synchronization of the initial cell-substratum contact and one to enable both a defined removal force to be exerted onto the cells for displacement of unbound cells and determination of the relative binding strengths of adhering cells. The fluorescently tagged cells are monitored in specifically devised, disposable microplate assemblies by a two-sided fluorescence detection through the computer-interfaced SPECTRAFLUOR microplate fluorometer. The assay is rapid, accurate, reproducible and adaptable to small numbers of delicate primary cells that can ideally be labeled with the fluorochrome calcein AM (or analogous vital fluorescent dyes). Most uniquely, CAFCA provides (i) means of assessing the precise number of cells bound to a given substratum out of the total amount of cells contained within the population to be analyzed and (ii) a means of establishing the attachment strengths (i.e., dynes/cell) in a high number of samples/conditions simultaneously. CAFCA is therefore expected to make a substantial methodological and conceptual contribution to the range of available assays aimed at examining cellular interactions in vitro and promises the potential of being able to transpose automated versions of these tests for routine use in laboratories.