Air bubbles Take the Stage in Flow Cytometric Drug Screening

capacity attributes, originating from the large number of different (mainly optical) signal channels (up to ~17 different colour fluorescence and light scattering signals) and the high speed of cell detection (~103-104/ sec). While slide based cytometry (laser scanning cytometry, LSC) is an excellent tool for monitoring adherent cells in their close to-native environments, for non-adherent cells in suspension, flow cytometry is the method of choice. Now, the problem at hand is how to convert a flow cytometer traditionally used in a „single sample-single run mode” into a device with wich different samples can be measured at a 40-100 samples/minute rate, i.e. to find the appropriate interface between an array of a huge number of different cell samples and the flow cytometer! The answer to this problem is given by the group of L. A. Sklar in New Mexico, in their works aiming at converting flow cytometry into a potent tool for drug discovery during the past decade. Experiencing initially with and 8-pole flow switch - „plug cytometry” [3] -, their work culminated in inventing a very ingenious sample „aspirating probe and transfer” arrangement called HyperCyt platform [4-6].


Introduction
Those who ever used a stream-in-air flow cytometer and when running their most interesting samples they just got an air bubble trapped in the nozzle never dreamed of a time when this awful situation just turned back showing its good face in helping to find the best drug for curing a disease! The following story is somewhat reminiscent of that of photobleaching, which was originally has been discovered as a pure nuisence reducing visibility of fluorescent samples, but with time it started to be used as a "kinetic ruler" for probing phenomena mediating relaxation of the excited states of dyes, leading to elaboration of methods like "photobleaching fluorescence resonance energy transfer" or "pbFRET" [1]. The central task of simultaneously probing thousands-millions of protein-protein, protein-DNA interactions, posed by a demand for solving biological complexity and problems of drug discovery has been tackled by introducing a highly versatile family of bisosensing technologies of outstandingly high speed, sensitivity and degree of multiplexation, collectively termed as "high throughput-high content (or capacity) screening assays, HT-HCS-A" [2]. Driven largely by the development of microscopy and imaging techniques (mainly confocal microscopy) different imaging solutions have been introduced such as the different plate-readers in biochemistry.
In the field of cell biology, the analogue candidates are the shaping of wells, and sample mixing by rotation to ensure sample homogenity [6,7].
In addition to that it can be conveniently fit to any flow cytometer, another remarkable feature of the HyperCyt platform is that it can be parallelized. In their work, B.S. Edwards, et al. [8] demonstrate the usage of HyperCyt when 4 probes connected to their respective cytometers are operated in tandem, allowing simultanous sampling from four parralel 384-well sections of a 1536-well plate taking only 11 minutes! By applying multiplexed bead and cell based assays, the authors demonstrate that also at these high sampling rates good quality data -as assessed by the "Z' A promising approach to drug screening is when the physical screening process is carried out on a preselected collection of drugs and receptors structurally fitting into a classification scheme constructed in-silico based on previous experience on the behaviour (whether agonist or antagonist) and chemical structure of drugs and receptors ("virtual screening"). After prefiltering by a "virtual screening" scheme, the "hit-rate" of finding drugs of the appropriate properties can be substantially increased, by >10-fold, as reported by the authors [9]. As opposed to measurement of ligand binding in equilibrium ("primary" or "endpoint" assays), time-kinetical recordings of ligand binding ("secondary" or "timed" assays) are also made possible by the HyperCyt system, during which cells are continually mixed ("incubated") with preformed coctail of reagents during the aspirating probe-cytometer sample delevery time.
More detailed binding characteristics of drugs and their inhibitors, two colours for FRET donor and acceptor, and another one or two colours for gating out the necessary cell population(s) [10].
In this case the number of samples is "exponentially growing" with the number of receptors involved in the proximity mapping, due to the need for the donor-only and acceptor-only samples in addition to the double-labeled ones, a typical system amenable for multiplexing. This sampling technology is also amenable for a larger degree of multiplexing by applying new types of light emitters with sharper emission spectra -the "hyperchromacity principle" [11]. A future marriage with the emerging nanocrystal, nanolaser, and plasmonics technologies seem to be promising in this respect [12]. For slide and imaging based screening platforms the optical resolution is also decisive. New types of light sourcese.g. time-ordered squeezed light from a nonlinear crystal, with a reduced Poisson-noise [13] -offering larger spatial resolution are good candidates for developing more sensitive biosensors.