Modified Flow Cytometer Research Articles

Size distribution analysis of microstickies treated by enzyme mixtures in papermaking whitewater

Microstickies present a formidable challenge for papermakers. Many strategies have been explored to control them. Enzyme treatment is a promising technology, but the mechanism of its action has not been determined, thus inhibiting further application of this new technology. This study investigated characteristics and size distributions of microstickies treated by esterase-cellulase mixtures. Determination of particle size and number was accomplished using a modified flow cytometer, which combined streaming capillary flow, laser-based particle size analysis and fluorescent dye tracing. The results showed that treatment of samples with enzyme mixtures induced size reduction of the larger microstickies. This effect was most dramatic for 1:1 ratios of esterase to cellulase. The treated particles were more stable than untreated ones. The smaller microstickies treated with some ratios of esterase and cellulase tended to aggregate over time. The behaviors of microstickies treated by enzyme mixtures were only slightly affected by temperature and shearing action. The surface physicochemical characteristics of these particles indicated that changes of these basic properties affected the whole whitewater system and resulted in a new equilibrium among all the particles.

Monitoring of Response and Resistance in Plasma of EGFR-Mutant Lung Cancer Using Droplet Digital PCR.

The identification of oncogenic driver mutations has led to the rapid rise of genotype-directed treatments. However, genetic analysis of tumors remains cumbersome and a morbid experience for patients. Noninvasive assessment of tumor genotype, so-called "liquid biopsy," such as plasma genotyping represents a potentially transformative tool. Here we describe a genotyping protocol of cell-free plasma DNA (cfDNA) using Droplet Digital™ PCR (ddPCR™). ddPCR emulsifies DNA into ~20,000 droplets in which PCR is performed to endpoint in each droplet for both mutant and wild-type DNA. Droplets are run through a modified flow cytometer where mutant and wild-type DNA emit different colored signals. The count of these signals upon Poisson distribution analysis allows sensitive quantification of allelic prevalence.

Characterizing the pressure field in a modified flow cytometer quartz flow cell: A combined measurement and model approach to validate the internal pressure

We incorporated an ultrasound transducer into a flow cytometer to activate microbubbles passing the laser interrogation zone (J. Acoust. Soc. Am. 126, 2954–2962, (2009)). This system allows high throughput recording of the volume oscillations of microbubbles, and has led to a new bubble dynamics model that incorporates shear thinning (Phys. Med. Biol. 58, 985–998 (2013)). Important parameters in the model include the ambient microbubble size, R0, driving pressure, PA, and the shell parameters χ and κ, the shell elasticity and viscosity, respectively. R0 is obtained by calibrating the cytometer. Pressure calibration is difficult because the flow channel width (<200µm) is too small to insert a hydrophone. The objective of this study was to develop a calibration method for a 20-cycle, 1 MHz transient pressure field. The pressure field propagating through the channel and into water was compared to a 3-D FEM model. After validation, the model was used to simulate the driving pressure as input for the bubble dynamics model, leaving only χ and κ variables. This approach was used to determine the mechanical properties for different bubbles (albumin, lipid, and lyzozyme shells). Excellent fits were obtained in many cases, but not all, suggesting heterogeneity in microbubble shell parameters.

Birth of Kids After Artificial Insemination with Sex‐Sorted, Frozen‐Thawed Goat Spermatozoa

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX(®) ) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n=2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25ml pellets on dry ice or 0.25ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p>0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3:1 (F:M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids.

A flow cytometer based protocol for quantitative analysis of bloom-forming cyanobacteria (Microcystis) in lake sediments

A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W × 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3×104 cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 μm in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium.

Microbubble sizing and shell characterization using flow cytometry

Experiments were performed to size, count, and obtain shell parameters for individual ultrasound contrast microbubbles using a modified flow cytometer. Light scattering was modeled using Mie theory, and applied to calibration beads to calibrate the system. The size distribution and population were measured directly from the flow cytometer. The shell parameters (shear modulus and shear viscosity) were quantified at different acoustic pressures (from 95 to 333 kPa) by fitting microbubble response data to a bubble dynamics model. The size distribution of the contrast agent microbubbles is consistent with manufacturer specifications. The shell shear viscosity increases with increasing equilibrium microbubble size, and decreases with increasing shear rate. The observed trends are independent of driving pressure amplitude. The shell elasticity does not vary with microbubble size. The results suggest that a modified flow cytometer can be an effective tool to characterize the physical properties of microbubbles, including size distribution, population, and shell parameters.

Factors influencing the “sortability” of stallion spermatozoa into X- and Y-chromosome bearing populations

Intrinsic differences between stallions exist for semen traits such as motility, morphology fertility and the ability of spermatozoa to survive cryopreservation processes. Ejaculates from 11 stallions were used to test the differences between stallions when selecting X- and Y-chromosome bearing spermatozoa using a modified flow cytometer. Data on orientation and viability of spermatozoa were collected during sex-sorting, and motility characteristics of sex-sorted and non-sorted (control) spermatozoa were assessed before and after cryopreservation. An index was created to rank each stallion in order of their suitability for sex-sorting using the data generated by the flow cytometry software. Motility of spermatozoa was higher after sorting and cooling than in the fresh ejaculates, but was significantly lower after thawing in comparison to fresh semen for both sex-sorted and non-sorted spermatozoa. Semen samples with a high percentage of food dye positive, defined as dead, spermatozoa had a low sortability index and ranking. Thus, percentage of dead spermatozoa in the semen sample was identified as the most important factor determining sortability. We conclude that variation between stallions exists for the sortability of their spermatozoa and that the sortability index is a useful tool for the selection of suitable stallions for a sex-sorting program.

Microenvironment-Controlled Encapsulation (MiCE) Process: Effects of PLGA Concentration, Flow Rate, and Collection Method on Microcapsule Size and Morphology

To evaluate the real-time effects of formulation and instrumental variables on microcapsule formation via natural jet segmentation, a new microencapsulation system termed the microenvironment-controlled encapsulation (MiCE) process was developed. A modified flow cytometer nozzle hydrodynamically focuses an inner drug and outer polymer solution emanating from a coaxial needle assembly into a two-layer compound jet. Poly(lactic-co-glycolic acid) (PLGA) dissolved in a water-miscible organic solvent resulted in formation of reservoir-type microcapsules by interfacial phase separation induced at the boundary between the PLGA solution and aqueous sheath. The MiCE process produced microcapsules with mean diameters ranging from 15-25 microm. The resultant microcapsule size distribution and number of drug cores existing within each microcapsule was largely influenced by the PLGA concentration and microcapsule collection method. Higher PLGA concentrations yielded higher mean diameters of single-core microcapsules. Higher drug solution flow rates increased the core size, while higher PLGA solution flow rates increased the PLGA film thickness. The MiCE microencapsulation process allows effective monitoring and control of the instrumental parameters affecting microcapsule production. However, the microcapsule collection method in this process needs to be further optimized to obtain microcapsules with desired morphologies, precise membrane thicknesses, high encapsulation efficiencies, and tight size distributions.

Photon-burst analysis in two-photon fluorescence excitation flow cytometry.

We studied the use of a dramatically reduced testing zone in combination with two-photon excitation and photon-burst analysis in high-throughput rare-event detection simulation using a modified flow cytometer. Two-photon excitation measurements were performed with a mode-locked titanium:sapphire laser. Fluorescence emission was measured with a photon-counting avalanche photodiode. Measured signal was analysed offline by autocorrelation and burst detection methods. Test samples were composed of full blood and orange fluorescent polystyrene nanospheres mixed in full blood. Results show that two-photon fluorescence excitation and time-correlation analysis provide a good signal-to-noise ratio for rare-event particle detection in a turbid sample environment.

Selective electrofusion of conjugated cells in flow

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.

Inability of Flow Cytometry to Distinguish X- from Y-Chromosome-Bearing Spermatozoa of Chinook Salmon

Abstract A modified flow cytometer was used to look for differences in DNA content between X- and Y-chro mosome-bearing spermatozoa of chinook salmon (Oncorhynchus tshawytscha). This system has been used successfully to make such determinations in several mammalian species, but it could neither distinguish between X- and Y-chromosome-bearing spermatozoa in sperm from heterogametic males, nor between total DNA content of sperm from heterogametic and homogametic (hormonally masculinized) males.

Very early detection of changes associated with cellular activation using a modified flow cytometer

A sample station modification previously described has been redesigned to provide greater flexibility and enhanced performance. The improved modification provides mixing and temperature regulation in a compact unit that mounts close to the nozzle holder for reduced transit times, allowing for addition of mediators to a sample in place on the flow cytometer, with observation of results in approximately 1 s. An electronic circuit activated at the time of injection generates full-scale pulses in the forward scatter channel. This provides the data with a time stamp for direct correlation of injection and cellular response. A detailed description of the modification, performance verification data, and practical applications in the measurements associated with cellular activation are presented.

A new principle of cell sorting by using selective electroporation in a modified flow cytometer

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 microseconds and resulting field strengths of 2.0 and 3.2 x 10(6) V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.

Attogram detection limit for aqueous dye samples by laser-induced fluorescence.

A modified flow cytometer has been used to detect attogram quantities of aqueous rhodamine 6G by laser-induced fluorescence analysis. A detection limit of 28 attograms (35,000 molecules) was obtained, nearly two orders of magnitude better than earlier measurements. The detection limit in concentration units was 1.4 x 10(-13) mole per liter. During the 1-second measurement period, the total volume sampled was 0.42 microliter. On average, only half a rhodamine 6G molecule was present in the 6-picoliter probed volume.