Cell Counting Device Research Articles

Possible induction of apoptotic and necrotic death pathways in lung cancer cells by Clinopodium vulgare L. and phenolic acids and flavonoids detection by LC-MS-MS

The objective of this research was to ascertain the phenolic content and antiproliferation and inducing of apoptosis activities of methanol extract of Clinopodium vulgare(CVME) on lung cancer cells.Initially, the viability and proliferation of lung cancer cells treated with CVME were assessed using MTT staining. AnnexinV/Propodium iodide fluorescent staining was then utilized to scan apoptotic cells with a cell counting device. Furthermore, the mRNA levels of proapoptotic Bax and antiapoptotic Bcl-2 were assessed via real-time PCR. Additionally, LC-MS-MS was used to determine the phenolic content of CVME. Following the administration of varying doses of CVME, it was found that the viability and proliferation of lung cancer cells reduced. The Bax and Bcl-2 mRNA levels did not significantly alter, however it was demonstrated that the cells killed by the necrotic pathway rather than by apoptosis. The main components of flavonoids and phenolic acids in CVME were found to be resveratrol and caffeic acid, respectively. Consequently, it can be said that lung cancer cells are inhibited by CVME, an abundant source of phenolics, via the necrotic pathway.

An automated approach for hemocytometer cell counting based on image-processing method

Evaluating cell viability plays an important role in routine cell culture, toxicology, and drug development. Trypan blue stain is one of the simplest assays that are used to determine the number of viable cells in a cell suspension. The stained cell suspension can then be counted under a microscope using a hemocytometer. Hemocytometers have a fixed volume, making it possible to calculate the concentration of cells in the sample and measure viability. Most of the related studies are conducted using manual counting methods. However, manual counting is time-consuming and laborious. While various automated cell counting devices have been proposed, they tend to be too expensive for widespread deployment. Although low-cost automated cell counting methods presented in the literatures provide high accuracy and cost-effective alternatives, only cells in the field of view of microscope with unknown volume are counted. This will make the concentration inestimable. Accordingly, the present study proposes a low-cost but accurate method for hemocytometer cell counting using an image-processing approach. The accuracy of the proposed method is demonstrated by comparing the cell counting results obtained for human cervical carcinoma (HeLa) cells with the manual counts. It is shown that the proposed approach has a mean absolute percentage error of less than 6.26 % for samples containing a mixture of live and dead cells. Moreover, the proposed system is equipped with a simple and intuitive human machine interface such that the counting task can be performed by individuals with no prior experience in the image-processing field.

Effects of storage duration on haematological parameters of the red eared slider - Trachemys Scripta Elegans

Reptiles, especially turtles, are becoming increasingly popular as pets. The haematological evaluation of turtles is an irreplaceable diagnostic tool in veterinary practice. However, the morphologic distinctiveness of turtle blood limits the use of electronic cell-counting devices, making time-consuming, manual counting techniques and evaluation of blood smears necessary. Many samples are dispatched to a laboratory over long distances, where a delay of 24 h or more may occur. At weekends, this interval may exceed 48 h. The objective of the present study was to determine the effect of storage duration at refrigerator temperature (4 °C) on the counts of red blood cells (RBC) and white blood cells (WBC), and on the mean corpuscular volume (MCV) and packed cell volume (PCV) in blood samples from healthy adult red-eared sliders. Blood samples were collected by venipuncture from the occipital venous sinus from six apparently healthy adult red-eared sliders, aged 2 to 4 years. Blood samples were analysed immediately after sampling to obtain the baseline value (BV) of the red blood cell count, white blood cell count and packed cell volume percentage. Blood was stored at 4 °C and the haematological analyses were performed after 24h, 48h and 72h. The results showed the same level of stability for RBC and WBC count, and MCV values during 72 hours of storage at 4 °C and for PCV during 48 hours. Handling of blood samples, and duration of storage of the blood samples can significantly influence the results/values of haematological tests. Consequently, the obtained values of the determined haematological parameters of improperly stored or handled blood samples can give a misleading interpretation of the results on the animal’s health status.

Estimation of Cell Number by Hemocytometry Counting.

The number of mammalian cells in a defined volume of medium can be measured using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. A hemocytometer contains two chambers, each of which when filled and coverslipped contains a total volume of 9 µL. Each chamber is ruled into nine major squares, and each square is 1 × 1 mm with a depth of 0.1 mm. Thus, when coverslipped, the volume of each square is 0.1 mm3 or 0.1 µL. Additional subdivisions of the major nine squares are not necessary for counting and can be ignored.

New inflammatory indicators: association of high eosinophil-to-lymphocyte ratio and low lymphocyte-to-monocyte ratio with smoking.

ObjectiveSmoking has been proven to increase systemic inflammation in previous studies using different biomarkers. The eosinophil-to-lymphocyte ratio (ELR), neutrophil-to-lymphocyte ratio (NLR), and lymphocyte-to-monocyte ratio (LMR) are new indicators of systemic inflammation that are used as predictors of systemic inflammation, morbidity, and mortality associated with many diseases. We investigated the effects of smoking on these inflammatory indicators.MethodsIn total, 616 consecutive smoking healthy subjects and 387 age-matched nonsmoking healthy subjects were enrolled. White blood cell counts (neutrophils, lymphocytes, basophils, eosinophils, and monocytes) were determined by electrical impedance with an automatic blood cell counting device. The ELR, LMR, and NLR were calculated based on these cell counts. Smoking habits of participants were calculated as pack/year.ResultsThe NLR and ELR were significantly higher and the LMR was significantly lower in smokers than nonsmokers. The pack-years were positively correlated with the NLR and ELR and negatively correlated with the LMR.ConclusionA high NLR and ELR and low LMR are associated with cigarette smoking and may be useful indicators of systemic inflammation activity, even in healthy smokers. Smokers with a high NLR and ELR and low LMR can easily be identified during routine blood analysis and might benefit from preventive treatment.

Application of NucleoCounter for the comprehensive assessment of murine cultured neurons during infection with Equine Herpesvirus type 1 (EHV-1).

The NucleoCounter NC-3000, a portable high-speed cell counting device based on the principle of fluorescence microscopy, provides the alternative method for standard flow cytometry. The main objective of the study was to apply an efficient technique for the assessment of the primary murine neurons culture infected with either neuropathogenic or non-neuropathogenic strains of Equine Herpesvirus type 1 (EHV-1). Using the NucleoCounter NC-3000 we have observed a decrease in mitochondrial potential and reduction in cells viability but we have not observed changes in the cell cycle of cultured neurons infected with all EHV-1 strains.

Determination of tonicity effects of ketoacids and lactate by use of two canine red blood cell assays.

To determine the tonicity effects of β-hydroxybutyrate, acetoacetate, and lactate in canine RBCs. RBCs from approximately 40 dogs. 2 in vitro methods were used to conduct 4 experiments. The modified osmotic fragility assay was used to measure the ability of ketoacid salts added to serial sucrose dilutions to protect RBCs from osmotic hemolysis. In a second assay, a handheld cell counting device was used to measure changes in RBC diameter to assess the tonicity effect of solutions of ketoacid and lactate salts. For the modified osmotic fragility assay, all ketoacid salts had an osmoprotective effect, but the effect was determined to be completely attributable to the tonicity effect of added cations (sodium and lithium) and not the ketoacid moieties. However, both the sodium and lithium lactate salts provided osmoprotection attributable to both the cation and lactate anion. For the second assay, RBC diameter was significantly increased with the addition of urea (an ineffective osmole) but did not change with the addition of glucose (an effective osmole), which established the behaviors of ineffective and effective osmoles in this assay. The RBC diameter was significantly increased over that of control samples by the addition of sodium β-hydroxybutyrate, lithium acetoacetate, and lithium lactate but was decreased by the addition of sodium lactate. For both assays, β-hydroxybutyrate and acetoacetate acted as ineffective osmoles, whereas lactate acted as an effective osmole in 3 of 4 experiments.

A simple and low-cost device performing blood cell counting based on lens-free shadow imaging technique

Complete blood count (CBC) is a basic and important diagnostic procedure in pathological laboratories. However, current technologies such as hemocytometry, impedancemetry, or flow-cytometry, are either time-consuming or requiring bulky and high-cost of instrumentation. To address this issue we demonstrate a compact, cost-effective, and automated blood cell counting method based on the lens-free shadow imaging technique. For that a lens-free shadow imaging technique based blood cell counting device was fabricated using a blue light emitting diode (LED) and a complementary metal oxide semiconductor (CMOS) image sensor. The shadow images of the blood cells, i.e., diffraction patterns, were captured by the device and processed automatically using a custom developed algorithm to count and differentiate the cells. The comparative study from 21 outpatient whole blood samples was carried out with the fully automated hematology analyzer (LH 750, Beckman Coulter). The comparison between the lens-free shadow imaging device and the standard reference system showed correlation indices of 0.878 for red blood cell (RBC) and 0.927 for white blood cell (WBC). The linearity comparison gave a statistical trend of y=0.9432x with R2=0.99 for RBC and y=0.7395x with R2=0.92 for WBC. Also the correlation coefficient for WBC subpopulations, i.e., neutrophil, lymphocyte, and monocyte, for each individual sample was >0.90. This cost-effective and compact blood cell counting system can be utilized as a powerful point of care (POC) tool, which could play an important role in primary healthcare, especially in areas with limited resources. Since the system is capable to send the images wirelessly and process them on a moderate smart phone, therefore, the proposed system possesses great potential in telemedicine applications.

Flow metering characterization within an electrical cell counting microfluidic device

Microfluidic devices based on the Coulter principle require a small aperture for cell counting. For applications using such cell counting devices, the volume of the sample also needs to be metered to determine the absolute cell count in a specific volume. Hence, integrated methods to characterize and meter the volume of a fluid are required in these microfluidic devices. Here, we present fluid flow characterization and electrically-based sample metering results of blood through a measurement channel with a cross-section of 15 μm × 15 μm (i.e. the Coulter aperture). Red blood cells in whole blood are lysed and the remaining fluid, consisting of leukocytes, erythrocyte cell lysate and various reagents, is flown at different flow rates through the measurement aperture. The change in impedance across the electrodes embedded in the counting channel shows a linear relationship with the increase in the fluid flow rate. We also show that the fluid volume can be determined by measuring the decrease in pulse width, and increase in number of cells as they pass through the counting channel per unit time.

Integrated blood cell counting device using a hydrophobic surface treatment

We fabricated an integrated blood cell counting system composed of planar blood preparation and blood cell counting devices. The planar blood preparation and counting devices were made of injection-molded polystyrene. 2 μL and 0.125 μL of whole blood were separated by hydrophobic passive valves and mixed with 50 μL of diluting fluid in the preparation device to accurately dilute the blood 25-fold and 400-fold, as primary and secondary dilution. We found that the hydrophobic fluoric resin surface treatments for the passive valves and the wall of the channels were effective for diluting whole blood. After the diluted blood was transferred into the blood counting devices, the number of blood cells was counted based on the change in electrode conductance at flow rate about 1 μL/s (50–500 cells/s). The coefficient of variation for the reproducibility of the blood cell counting was <2%, which is a desirable value for clinical use.

Validation of analytical methods in GMP: the disposable Fast Read 102® device, an alternative practical approach for cell counting

BackgroundThe quality and safety of advanced therapy products must be maintained throughout their production and quality control cycle to ensure their final use in patients. We validated the cell count method according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and European Pharmacopoeia, considering the tests’ accuracy, precision, repeatability, linearity and range.MethodsAs the cell count is a potency test, we checked accuracy, precision, and linearity, according to ICH Q2. Briefly our experimental approach was first to evaluate the accuracy of Fast Read 102® compared to the Bürker chamber. Once the accuracy of the alternative method was demonstrated, we checked the precision and linearity test only using Fast Read 102®. The data were statistically analyzed by average, standard deviation and coefficient of variation percentages inter and intra operator.ResultsAll the tests performed met the established acceptance criteria of a coefficient of variation of less than ten percent. For the cell count, the precision reached by each operator had a coefficient of variation of less than ten percent (total cells) and under five percent (viable cells). The best range of dilution, to obtain a slope line value very similar to 1, was between 1:8 and 1:128.ConclusionsOur data demonstrated that the Fast Read 102® count method is accurate, precise and ensures the linearity of the results obtained in a range of cell dilution. Under our standard method procedures, this assay may thus be considered a good quality control method for the cell count as a batch release quality control test. Moreover, the Fast Read 102® chamber is a plastic, disposable device that allows a number of samples to be counted in the same chamber. Last but not least, it overcomes the problem of chamber washing after use and so allows a cell count in a clean environment such as that in a Cell Factory. In a good manufacturing practice setting the disposable cell counting devices will allow a single use of the count chamber they can then be thrown away, thus avoiding the waste disposal of vital dye (e.g. Trypan Blue) or lysing solution (e.g. Tuerk solution).

Comparison of methods for assessing integrity of equine sperm membranes

Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50–79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = −3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = −11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed(1) who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate(2). To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry(1). For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments(1). The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection(3) in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2. To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1. The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

Utility of NucleoCounter for the chondrocyte count in the collagenase digest of human native cartilage

In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R(2)=0.9999) and reproducibility (%CV: 2.00-8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R(2)=0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20-1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions.

Automated Handheld Instrument Improves Counting Precision Across Multiple Cell Lines

Counting cells is often a necessary but tedious step for in vitro cell culture. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, a survey (1) of 400 researchers who count cells revealed that 71% use a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse that results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of error in hemocytometry include uneven cell distribution in the sample; too many or too few cells in the sample; subjective decisions as to whether a given cell falls within the defined counting area; contamination of the hemocytometer; user-to-user variation; and variation of hemocytometer filling rate (2). To alleviate the tedium associated with manual counting, 29% of the researchers in the survey count cells using automated cell counting devices; these include vision-based counters—systems that detect cells using the Coulter principle—and flow cytometers (1). For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments (1). The ScepterTM cell counter (Millipore) combines the ease of automated instrumentation and the accuracy of Coulter impedance-based particle detection (3) in an affordable, handheld format (Figure 1). The instrument, which is the size of a pipet, uses a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip (Figure 1, inset) has a microfabricated, cell-sensing zone that can discriminate cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter graphically displays cell population statistics as histograms. Counting is typically completed in less than 20 seconds.

Individual cells immobilization for water-borne pathogen detection and enumeration

A cell counting device has been proposed and implemented for water-borne pathogen detection for drinking water quality monitoring applications. Our approach is based on magnetically-labelled cells immobilization in a high density array of individual cell for optical cell counting. The device has been tested for two water-borne pathogens: Giardia Lamblia & Cryptosporidium. An individual cell immobilization efficiency of 82% was achieved.

Microfluidic cell counter with embedded optical fibers fabricated by femtosecond laser ablation and anodic bonding

A simple fabrication technique to create all silicon/glass microfluidic devices is demonstrated using femtosecond laser ablation and anodic bonding. In a first application, we constructed a cell counting device based on small angle light scattering. The counter featured embedded optical fibers for multiangle excitation and detection of scattered light and/or fluorescence. The performance of the microfluidic cell counter was benchmarked against a commercial fluorescence-activated cell sorter.

Use of automated imaging and analysis technology for the detection of aneuploidy in human sperm

To determine the precision and accuracy of an automated cell counting system when applied to counting aneuploidies in sperm samples. Prospective pilot study. Andrology clinic and research laboratory in a university teaching hospital. Ten anonymous sperm donors of known fertility and two patients seeking infertility treatment. Semen samples were processed for detection of aneuploidies for chromosomes 13, 18, 21, X, and Y with use of fluorescent in situ hybridization. The detection of chromosome aneuploidy was performed both by manual counting and by the use of an automated cell counting system with manual review of aneuploid sperm. Semen samples were judged for the percent aneuploidy for chromosomes 13, 18, 21, X, and Y when counted manually or with the use of the automated cell counting system and review by a technician. The sperm aneuploidy rates determined by the automated cell counting system and careful review were comparable with those obtained by manual counting by a trained technician. These preliminary data demonstrate that automated cell counting devices may be useful in increasing productivity in aneuploidy detection in sperm and may become an alternative to the labor-intensive manual counting by technicians.

A Microchip Approach for Practical Label-Free CD4+ T-Cell Counting of HIV-Infected Subjects in Resource-Poor Settings

Simple affordable CD4 cell counting is urgently needed to stage and monitor HIV-infected patients in resource-limited settings. To address the limitations of current approaches, we designed a simple, label-free, and cost-effective CD4 cell counting device using microfluidic technology. We previously described the fabrication of a microfluidic system for high-efficiency isolation of pure populations of CD4+ T cells based on cell affinity chromatography operated under controlled flow. Here, we compare the performance of a microfluidic CD4 cell counting device against standard flow cytometry in 49 HIV-positive subjects over a wide range of absolute CD4 cell counts. We observed a close correlation between CD4 cell counts from the microchip device and measurements by flow cytometry, using unprocessed whole blood from HIV-positive adult subjects. Sensitivities for distinguishing clinically relevant thresholds of 200, 350, and 500 cells/microL are 0.86, 0.90, and 0.97, respectively. Specificity is 0.94 or higher at all thresholds. This device can serve as a functional cartridge for fast, accurate, affordable, and simple CD4 cell counting in resource-limited settings.