Assessment of cell viability.

Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre-fixation viability of fixed cells either before or after fixation with amine-reactive dyes suitable for a range of excitation wavelengths. Membrane-impermeable dead cell and live cell dyes as well as dye-exclusion procedures for microscopy are also included.

Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells.

All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required.

Sensitivity measurement and compensation in spectral imaging.

Background: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented.

Methods: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer.

Cutting edge: FADD is not required for antigen receptor-mediated NF-kappaB activation.

Recently, it has been demonstrated that stimulated T cells bearing defects in caspase-8 fail to promote nuclear shuttling of NF-kappaB complexes. Such cells display strikingly similar proliferative and survival defects as T cells lacking Fas-associated death domain protein (FADD) function. We characterized NF-kappaB signaling in T cells bearing a dominant-negative FADD transgene (FADDdd).

Precise and rapid assessment of Escherichia coli adherence to vaginal epithelial cells by flow cytometry.

Background: In the pathogenesis of Escherichia coli urinary tract infections (UTIs) in women, infecting bacteria adhere to vaginal and periurethral epithelial cells prior to ascending to the bladder and causing infection. Complex interactions among specific bacterial adhesins and various host factors appear to influence adherence of E. coli to mucosal surfaces such as the urogenital epithelium.

Multiparameter cytokine-specific affinity matrix assay for the determination of frequencies and phenotype of antigen-reactive T cells.

A major challenge in Phase I clinical trials of cancer vaccines is how to identify the optimal regimen and/or dosing for Phase II and III trials. Immunologic responses to tumor-associated antigens (TAA) could potentially serve as surrogate markers. Assays which detect cytokine production and/or secretion by antigen (Ag)-reactive T cells are increasingly being used to monitor such responses in clinical trials.