23 research outputs found
Ca2+ signaling modulates cytolytic T lymphocyte effector functions
Cytolytic T cells use two mechanisms to kill virally infected cells, tumor cells, or other potentially autoreactive T cells in short-term in vitro assays. The perforin/granule exocytosis mechanism uses preformed cytolytic granules that are delivered to the target cell to induce apoptosis and eventual lysis. FasL/Fas (CD95 ligand/CD95)–mediated cytolysis requires de novo protein synthesis of FasL by the CTL and the presence of the death receptor Fas on the target cell to induce apoptosis. Using a CD8+ CTL clone that kills via both the perforin/granule exocytosis and FasL/Fas mechanisms, and a clone that kills via the FasL/Fas mechanism only, we have examined the requirement of intra- and extracellular Ca2+ in TCR-triggered cytolytic effector function. These two clones, a panel of Ca2+ antagonists, and agonists were used to determine that a large biphasic increase in intracellular calcium concentration, characterized by release of Ca2+ from intracellular stores followed by a sustained influx of extracellular Ca2+, is required for perforin/granule exocytosis. Only the sustained influx of extracellular Ca2+ is required for FasL induction and killing. Thapsigargin, at low concentrations, induces this small but sustained increase in [Ca2+]i and selectively induces FasL/Fas-mediated cytolysis but not granule exocytosis. These results further define the role of Ca2+ in perforin and FasL/Fas killing and demonstrate that differential Ca2+ signaling can modulate T cell effector functions
Rotation-Driven Microfluidic Disc for White Blood Cell Enumeration Using Magnetic Bead Aggregation
We recently defined a magnetic bead-based
assay that exploited
an agglutination-like response for DNA and applied it to DNA-containing
cell enumeration using inexpensive benchtop hardware [J. Am. Chem. Soc. 2012, 134 (12), 5689−96]. Although cost-efficient, the open-well format
assay required numerous manual steps, and the magnetic field actuation
scheme was not readily adaptable for integration. Here, we demonstrate
a low-cost (<$2 in-lab), higher-throughput “pinwheel assay”
platform that relies on a combination of a disposable rotation-driven
microdisc (RDM), and a simple bidirectional rotating magnetic field
(bi-RMF). The assay was transformed into an integrated microfluidic
system using a multilayered polyester microfluidic disc created through
laser print, cut and laminate fabrication, with fluid flow controlled
by rotation speed without any mechanical valves. The RDM accepts four
samples that undergo on-chip dilution to five different concentrations
that cover the effective concentration range needed for downstream
cell counting by pinwheel assay. We show that a bi-RMF is effective
for the simultaneous actuation of pinwheel assays in 20 detection
chambers. The optimization of the bi-RMF frequencies allows the RDM-based
pinwheel assay detect human genomic DNA down to a mass of human genomic
DNA (5.5 picograms) that is roughly equal to the mass in a single
cell. For proof of principle, enumeration of the white blood cells
in human blood samples on the RDM provided data correlating well (C.V.
of 10%) with those obtained in a clinical lab. Fusing the cost-effective
RDM with a simple bi-RMF provides a promising strategy for automation
and multiplexing of magnetic particle-based agglutination assays
Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads
A novel solid phase extraction technique is described where DNA is bound and eluted from magnetic silica beads in a manner where efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the beta-globin gene. By effectively controlling the movement of the solid phase in the presence of a static sample, the issues associated with reproducibly packing a solid phase in a microchannel and maintaining consistent flow rates are eliminated. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 0.6 mu L of blood at a concentration which is suitable for PCR amplification. In addition, the technique presented here requires inexpensive, common laboratory equipment, making it easily adopted for both clinical point-of-care applications and on-site forensic sample analysis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2005/04473-4
Label-Free Method for Cell Counting in Crude Biological Samples via Paramagnetic Bead Aggregation
Under
chaotropic conditions, DNA released from lysed cells causes
the aggregation of paramagnetic beads in a rotating magnetic field
in a manner that is independent of the presence of other cellular
components. The extent of aggregation correlates with the mass of
DNA in a quantitative manner (Leslie, D. C. et al., <i>J. Am.
Chem. Soc</i>. <b>2012</b>, <i>134</i>, 5689–96),
and from this, the number of DNA-containing cells in the sample can
be enumerated. Microbial growth testing is demonstrated by monitoring
bead aggregation with E. coli in the
presence of ampicillin. Without the need for fluorescent labeling
or Coulter counting, the white
blood cell count can be defined directly from a microliter of crude
whole blood. Specificity is brought to the process by coupling bead-based
immunocapture with DNA–bead aggregation allowing for the enumeration
of CD4+ T cells from human blood samples. The results of DNA-induced
bead aggregation had a 95% correlation with those generated by flow
cytometry. With the process requiring only inexpensive, widely available
benchtop laboratory hardware, a digital camera, and a simple algorithm,
this provided a highly accessible alternative to more expensive cell-counting
techniques