An elution—extrusion countercurrent chromatography workstation was designed and built in-house for fractionating crude natural product extracts. The engineering efforts of this project included both custom hardware and software integration. The resulting workstation operates four individual chromatography coils for natural product extract separation and purification. The workstation offers the ability to collect fractions into 16 × 100 mm borosilicate glass test tubes or allows for stream splitting of the coil effluent so that the fractions can be collected into both 16 × 100 mm borosilicate test tubes and a 96-well microplate. Solvent pumping for each of the chromatography columns is achieved through the use of syringe pumps. The workstation is controlled though a custom control software application, C_Cubed, written in Visual Basic 6.0 (VB6). Software architecture consists of three levels. At the lowest level, there are ActiveX dll device drivers that interface with the physical hardware. The middle layer is a custom scheduler allowing for multitasking of instrument movements. The upper level is composed of a Wizard-style user interface that mimics the scientific workflow. Finally, by using a software timer the software application was written to operate on a single thread, but exhibit multithreaded behavior.

Utilizing flow cytometry, we previously demonstrated that the potassium channel blocker margatoxin (MgTX) inhibits the [Ca2+]i transient involved in T-cell activation. We wished to extend these studies to single-cell transients using florescence digital-imaging microscopy (DIM). However, the most currently available temperature-regulation chambers reuse part or all of the apparatus and introduce compounds via perfusion. Thus, these apparatuses are not suitable for studies involving compounds that are particularly sticky. We have designed a dual-temperature regulation system that will maintain Nunc, eight-well, coverglass-bottom, disposable chambers, and three disposable addition pipets at 37 degrees C for physiological studies on an inverted digital-imaging microscope. We have demonstrated that calcium transients of human T lymphocytes can be initiated and monitored reproducibly during the addition of three distinct chemical species. The DIM results correlate with flow cytometry measurements in the number of responding cells and the heterogeneity of the response in both control and MgTX-inhibited cultures. Additionally, DIM revealed that the [Ca2+]i transient is more rapid than the flow-cytometric measurement indicated. The correlation between flow cytometry and DIM permits the amalgamation of these results in the interpretation of studies on the regulation of T-cell activation.