Labeling Steps Research Articles

Establishment of a direct quantitative method for measurement of microRNA-224 in serum by UHPLC/MS/MS

MicroRNAs (miRNAs) are known as potential noninvasive biomarkers for cancer diagnosis. Previous studies have been reported that miR-224 is upregulated in hepatocellular carcinoma (HCC) tissues and sera samples. However, current available methods of miRNA detection typically require pre-enrichment, amplification and labeling steps, and mostly they are semi-quantitative. Herein, we developed an isotope dilution mass spectrometry approach to convert the signal of miR-224 into the mass response of a reporter peptide. Specifically, the newly formed DNA-peptide probe was hybridized with miR-224, which was biotinylated and attached to streptavidin agarose in advance. After through trypsinization, solid phase extraction and blow drying, it used a UHPLC/MS/MS-based quasi-targeted proteomics assay for miR-224 quantification and determines the peak area or peak height ratio of labeled and non-labeled analytes to which an isotope label was added to estimate the concentration of the analyte in the sample. Moreover, this method showed good linearity, precision, accuracy and recovery in the calibration range. In addition, it also demonstrated good performance in comparison with qRT-PCR. Taken together, this study may offer a novel direct quantitative method for serum miRNA analysis applied in clinical practice.

Fully automated integrated process for depletion of T cells with or without subsequent purification of NK cells from apheresis using the CliniMACS Prodigy

Background & Aim Donor-derived natural killer (NK) cells mediate graft-versus-leukemia effects low risk in inducing alloreactive graft-versus-host-disease and are used as component of T cell-depleted donor lymphocyte infusion (DLI) or purified. As clinical grade purification of NK cells requires two semi-automated separation procedures, including multiple manual hands-on steps, we developed an integrated fully automated process enabling either preparation of T cell-depleted DLI or clinical grade purification of NK cells within a single automated process on the CliniMACS Prodigy. Methods, Results & Conclusion The novel integrated CliniMACS Prodigy LP-3-56 System uses the CliniMACS Prodigy Tubing Set 320 for an fully automated procedure with integrated labeling and washing steps for both, CD3 and CD56 separation enabled through an integrated lytic rinsing sequence allowing subsequent CD56 enrichment in the same tubing set. By users choice the depletion of CD3+ cells will be performed with or without subsequent enrichment of CD56+ cells. For both strategies target cells are prepared with user-chosen formulation solution (e. g. sodium chloride or NK MACS medium). The new application software effectively depletes 9.6 × 109 CD3+ cells, and enriches 4.5 × 109 CD56+ cells from up to 40 × 109 total white blood cells. Inhouse evaluation runs with leukapheresis products (n= 29 in total, n=17 with CD3 depletion, n=12 with CD3 depletion and CD56 enrichment) were performed. After CD3 depletion, mean log depletion of T cells was 4.0. The mean recovery of NK cells was 89% with an average NK:T ratio-increase of 9509 fold. After CD3 depletion and CD56 enrichment, mean log depletion of T and B cells was 4.2 and 2.7, respectively. Purity of NK cells was 90.7% on average with a mean recovery of 49%. The majority of non-NK cells after the process were monocytes with a mean frequency of 5.9%. Functionality of purified NK cells was proven by their ability to lyse AML cells, to secrete cytokines after co-culture, and to perform antibody dependent cell-mediated cytotoxicity (ADCC) against Raji cells, using human CD20 antibody. In summary, the integrated fully automated CliniMACS Prodigy LP-3-56 System is capable of efficiently deplete CD3+ cells with or without subsequent CD56 enrichment to purify functional NK cells from leukapheresis products and provides target cells in formulation solution. The performance verification and submission to an European notified body for CE mark are the important next steps.

Milk Allergen Detection: Sensitive Label-Free Voltammetric Immunosensor Based on Electropolymerization

We present in this work, first time in literature, a voltammetric label-free biosensor for detection of alpha lactalbumin (α-LB) in meal samples. Food allergy concern necessitates strict regulations in food labelling and innovative monitoring strategies for prevention of sepsis-related death. As major food allergens, dietary samples often contain whey proteins, including α-LB as one of the key allergenic. Available detection methods for α-LB are based on successive labelling steps and hence have long assay times with limited sensitivity. The new detection method presented here is based on capturing of α-LB via entrapped α-LB antibody (α-LB-Ab) through electropolymerization of polypyrrole (PPy) and then measuring the conductivity decrease by differential pulse voltammetry (DPV). This new label-free voltammetric biosensor for α-LB exhibits the best limit of detection (0.19 fg/mL) published so far in literature, with high selectivity and capability to quantify α-LB in real milk samples too.

SiRNA-directed self-assembled quantum dot biosensor for simultaneous detection of multiple microRNAs at the single-particle level

MicroRNAs are essential post-transcriptional regulators and may act as the noninvasive biomarkers for disease diagnosis. Sensitive and multiplexed detection of microRNAs may facilitate the accurate and early clinical diagnosis, but the available methods are usually compromised by using organic dyes as the signal probes, laborious chemical and enzymatic manipulations, and the complicated reaction schemes. Here we reported a siRNA-directed self-assembled quantum dot (QD) biosensor for facile and simultaneous detection of multiple microRNAs. In this biosensor, the binding of microRNA targets with corresponding QD nanoprobes leads to the formation of siRNA duplexes, which not only induces the spectrally resolved coding of microRNAs, but also facilitates the assembly of QDs:magnetic nanoparticle bioconjugates for the isolation and enrichment of target microRNAs. The disassembled QDs can be sensitively detected by single-molecule detection, enabling quantitatively sensing of microRNAs at the single-particle level. This biosensor employs only QDs as the signal reporters, which can simultaneously detect multiple microRNAs from the same sample and achieves femtomolar sensitivity and single-base mismatch selectivity without the involvement of any target labeling and amplification steps. Moreover, it can be successfully applied for simultaneous detection of circulating microRNAs in clinical serum samples, holding great potential in non-invasive early diagnosis and biomedical researches.

Hierarchically Labeled Database Indexing Allows Scalable Characterization of Microbiomes.

SummaryIncreasingly available microbial reference data allow interpreting the composition and function of previously uncharacterized microbial communities in detail, via high-throughput sequencing analysis. However, efficient methods for read classification are required when the best database matches for short sequence reads are often shared among multiple reference sequences. Here, we take advantage of the fact that microbial sequences can be annotated relative to established tree structures, and we develop a highly scalable read classifier, PRROMenade, by enhancing the generalized Burrows-Wheeler transform with a labeling step to directly assign reads to the corresponding lowest taxonomic unit in an annotation tree. PRROMenade solves the multi-matching problem while allowing fast variable-size sequence classification for phylogenetic or functional annotation. Our simulations with 5% added differences from reference indicated only 1.5% error rate for PRROMenade functional classification. On metatranscriptomic data PRROMenade highlighted biologically relevant functional pathways related to diet-induced changes in the human gut microbiome.

New trends in single-molecule bioanalytical detection

Single-molecule sensing is becoming a major driver in biomarker assays as it is foreseen to enable precision medicine to enter into everyday clinical practice. However, among the single-molecule detection methods proposed so far, only a few are fully exploitable for the ultrasensitive label-free assay of biofluids. Firstly introduced single-molecule sensing platforms encompass low-background-noise fluorescent microscopy as well as plasmonic and electrical nanotransducers; these are generally able to sense at the nanomolar concentration level or higher. Label-based single-molecule technologies relying on optical transduction and microbeads that can scavenge and detect a few biomarkers in the bulk of real biofluids, reaching ultralow detection limits, have been recently commercialized. These assays, thanks to the extremely high sensitivity and convenient handling, are new trends in the field as they are paving the way to a revolution in early diagnostics. Very recently, another new trend is the label-free, organic bioelectronic electrolyte-gated large transistors that can potentially be produced by means of large-area low-cost technologies and have been proven capable to detect a protein at the physical limit in real bovine serum. This article offers a bird’s-eye view on some of the more significant single-molecule bioanalytical technologies and highlights their sensing principles and figures-of-merit such as limit of detection, need for a labelling step, and possibility to operate, also as an array, directly in real biofluids. We also discuss the new trend towards single-molecule proof-of-principle extremely sensitive technologies that can detect a protein at the zeptomolar concentration level involving label-free devices that potentially offer low-cost production and easy scalability.

Non‑invasive proteome‑wide quantification of skin barrier‑related proteins using label‑free LC‑MS/MS analysis.

A number of epidermal proteins are closely related to skin barrier function, the abnormalities of which can lead to specific skin diseases. These proteins must be quantified to further investigate the changes in the skin barrier between healthy and disease states. However, the non-invasive and proteome-wide quantification of skin proteins without any labelling steps remains a challenge. In this study, 3M medical adhesive tapes were used to obtain skin samples from volunteers. Proteins were extracted from fresh skin samples and digested with trypsin. Each tryptic peptide was analysed in three replicates using liquid chromatography with tandem mass spectrometry analysis and label-free quantification. The data were searched against the Human Universal Protein Resource (UniProt) to match with known proteins. Using this method, 1,157 skin proteins recorded in the UniProt were quantified. A total of 50 identical proteins were identified in the three replicate analyses of all samples with no significant differences in abundance. The results provided an objective metric for further study of skin ageing and various skin diseases. Specifically, the non-invasive proteome-wide method used in this study can be applied to future studies of skin diseases related to barrier destruction by monitoring the changes in the levels of epidermal proteins.

Architecting of a biodevice based on a screen-printed carbon electrode modified with the NiONP nanolayer and aptamer in BCM-7 detection

In this study, an ultrasensitive and robust biodevice implemented on a screen-printed carbon electrode (SPCE) surface is introduced. The β-casomorphin-7 (BCM-7) peptide as an early warning sign of autism disorder is distinguished in this system. The SPCE surface was directly electrodeposited with a nanolayer of the nickel oxide nanoparticles (NiONP). In next step, an Apt sequence as a capture of the BCM-7 was strongly attached on this surface. The embedded sensing interface offered some admirable characterizes to detect the BCM-7. The obtained DPV signals were reversely proportional to the concentrations of the BCM-7 through a stable binding reaction in two working linear ranges from 0.5 × 10−9-1.5 μmol l-. Also, an unrivaled limit of detection (LOD) value of 166.6 aM was achieved that is so superior by other reported methods in the BCM-7 sensing. This hand-held biodevice was satisfactorily tested for the BCM-7 detection in human urine and blood sample with an average recovery rate of ∼101.87 %. More importantly, this strategy is free from labeling steps, complex sample processing and interference from common biomolecules in blood or urine. Due to the inherent advantages of the SPCE and the NiONP, utilizing this facile sensing interface may be an ideal choice in constructing of the ultrasensitive biodevice with low cost for distinguishing of the autism disorder.

Titanium Carbide MXenes Mediated In Situ Reduction Allows Label-Free and Visualized Nanoplasmonic Sensing of Silver Ions.

The label-free assay has drawn extensive attention because it does not require a labeling step and enables direct interaction and signal transduction between the sensing unit and target analytes. Herein, we demonstrate a proof-of-principle concept of a label-free and visualized nanoplasmonic strategy for silver ions sensing, where only Ti3C2 MXenes are employed by exploring their excellent adsorption affinity and reductive property toward metal ions. Ag+ was adsorbed onto the surface of Ti3C2 MXene nanosheets, followed by the Ti3C2 MXenes mediated in situ silver nanoparticles (Ag NPs) generation without adding any extra stabilizing or reducing agent. The excellent localized surface plasmon resonances at a particular wavelength provide Ag NPs the capability for colorimetric assay with a detection limit of 0.615 μM. With the assistance of a smartphone, RGB analysis exhibited visualized results consistent with the results measured on a UV-vis spectrometer, promising a budget, simple-operating on-site detection. Moreover, the detection of Ag+ in real samples was achieved with satisfactory results meeting the analysis demand for the Drinking Water Standards of the World Health Organization (WHO) and the United States Environmental Protection Agency (U.S. EPA). These results reveal that Ti3C2 MXenes possess great potential in building convenient label-free colorimetry nanoplatforms and may evoke more inspirations to explore strategies for the direct sensing of analytes.

Computational techniques to segment and classify lumbar compression fractures.

Vertebral fractures are important indicators of osteoporosis. Fractures with partial collapse of vertebral bodies are referred to as vertebral compression fractures (VCFs) that are usually non-traumatic in nature. Some common causes of VCFs are trauma, bone failure related to osteoporosis (benign) and metastatic cancer (malignant). This paper aims at developing a system for computer-aided diagnosis to help in the detection, labeling and segmentation of lumbar vertebral body (VB) and to further classify each VB into normal, malignant and benign VCFs. After the initial preprocessing, morphological, shape and angular features are used in the detection, labeling and segmentation steps. Various shape and statistical texture features are extracted from the segmented VB and are fed to the classifier for the final decision. The segmentation and classification results obtained were compared with the ground truth manual segmentation of the lumbar VB and the decision labels of the fractures provided by the experts. The dice similarity coefficient (DSC) for segmentation reached up to 94.27%, and the classification results show that shape and texture features together are able to correctly classify with an accuracy rate of 95.34%. The final outcomes are expected to be useful in the analysis of vertebral compression fractures.

Assessment of instructions on panelist cognitive framework and free sorting task results: A case study of cold brew coffee

As the free sorting task becomes more common in sensory science, methodological research is needed to determine best practices for the test. Previous studies have shown that asking attribute-specific, “analytical” questions can bias the answers participants give to later “holistic” questions in a survey. For the free sorting task, this has led researchers to recommend only asking subjects to label or describe groups of products after they have finished sorting, but this approach is not based on empirical research. The present study investigated whether the separation of sorting and labeling steps is necessary by having 80 panelists sort 12 cold brew coffee samples in 3 conditions: labeling and describing the groups afterwards, at the same time, or not at all. Results were analyzed with DISTATIS, Hierarchical DISTATIS, and permutation tests. This work is novel in investigating the effect of labeling instructions on sorting results and provides some interesting insight into sensory impact of coffee bean parameters on cold brew coffees. No configurational differences were found based on the timing of labeling instructions and 38% of panelists labeled their groups without being told, suggesting future researchers may request that subjects label when convenient. While the most distinctive samples were the dark roast coffees, a large subgroup (n = 36) sorted the coffee samples partially based on country of origin. Given the lack of effect from different sorting instructions and the tendency for spontaneous, simultaneous labeling, simultaneous labeling is suggested as best practice for future sorting tasks.

High-Throughput Screening Using UHPLC-MS To Characterize the Subsite Specificities of Chitosanases or Chitinases.

Partially acetylated chitosan oligosaccharides (paCOS), consisting of β-1,4-linked N-acetyl-d-glucosamine and d-glucosamine units, possess diverse bioactivities that can be used for applications in, e.g., biomedicine, agriculture, and pharmaceutics. Establishing structure-function relationships and revealing modes of action requires the availability of structurally defined paCOS that can best be produced using chitin- and chitosan-modifying enzymes, such as chitinases and chitosanases, with known and defined subsite specificities. To enlarge the spectrum of such enzymes and, consequently, defined paCOS available, we have developed a two-step, microtiter plate-based high-throughput screening assay that allows quantification of the activity and subsite specificities of chitosan hydrolases. In a first step, the activities of the enzymes are quantified using a reducing end assay, and enzymes with sufficient activity are then screened for their subsite specificities using mass spectrometric analysis of their products when acting on well-defined chitosan polymers as substrates. The rapid UHPLC-ELSD-ESI-MS2 method does not require labeling steps or addition of standards, and the principal component analysis of the fragment ion intensities of just two isomeric oligomer groups, GlcNAc1GlcN3 and GlcNAc2GlcN2, sufficed to identify, in a directed evolution, the site-saturation mutagenesis library of Bacillus sp. MN chitosanase consisting of 167 muteins, enzymes that significantly differed in their subsite specificities from the wildtype enzyme. Detailed analyses of a few selected muteins proved that the screening method is efficient and accurate in predicting altered subsite specificities.

Label-free impedance flow cytometry for nanotoxicity screening

The development of reliable and cost-efficient methods to assess the toxicity of nanomaterials (NMs) is critical for the proper identification of their impact on human health and for ensuring a safe progress of nanotechnology. In this study, we investigated the reliability and applicability of label-free impedance flow cytometry (IFC) for in vitro nanotoxicity screening, which avoids time-consuming labelling steps and minimizes possible NM-induced interferences. U937 human lymphoma cells were exposed for 24 h to eight different nanomaterials at five concentrations (2, 10, 20, 50, and 100 μg/mL). The NMs’ effect on viability was measured using IFC and the results were compared to those obtained by trypan blue (TB) dye exclusion and conventional flow cytometry (FC). To discriminate viable from necrotic cells, the IFC measurement settings regarding signal trigger level and frequency, as well as the buffer composition, were optimised. A clear discrimination between viable and necrotic cells was obtained at 6 MHz in a sucrose-based measurement buffer. Nanomaterial-induced interferences were not detected for IFC. The IFC and TB assay results were in accordance for all NMs. The IFC was found to be robust, reliable and less prone to interferences due to the advantage of being label-free.

Highly sensitive impedimetric glycosensor for the determination of a ricin surrogate, Ricinus communis agglutinin I (RCA120)

A highly sensitive impedimetric glycosensor has been developed for the label-free detection of Ricinus communis Agglutinin I (RCA120) - a surrogate of potential security threat toxin ricin. In order to accomplish strong binding of RCA120 onto the glycosensor surface, the coverage of self-assembled monolayers of thiolated β-galactose derivative on gold electrode was optimized with varying ratios of thiolated ethylene glycol derivative. The association constant between the RCA120 and the β-galactose derivative with 10% coverage on gold electrode surface has been determined to be ca. 1.54 × 109 M−1, implying that the β-galactose derivative can be used as an effective recognition element for RCA120. The increase in electron transfer resistance through the selective binding of RCA120 onto the glycosensor measured by electron impedance spectroscopy was directly dependent upon the amount of RCA120 in sample solution. The present glycosensor can detect RCA120 from 8.33 × 10−11 M to 4.17 × 10−6 M with a limit of detection (S/N = 3) of 1.23 × 10−11 M, which is much lower than those of the previous reports. Since the proposed glycosensor does not need time-consuming labeling or signal amplification steps commonly carried out in sandwich-type RCA120 immunoassays, it provides the potential for a simple and rapid detection of a security threat toxin ricin.

Evaluation of acoustic-based particle separation methods

Purpose This paper aims to Separation and sorting of biological cells is desirable in many applications for analyzing cell properties, such as disease diagnostics, drugs delivery, chemical processing and therapeutics. Design/methodology/approach Acoustic energy-based bioparticle separation is a simple, viable, bio-compatible and contact-less technique using, which can separate the bioparticles based on their density and size, with-out labeling the sample particles. Findings Conventionally available bioparticle separation techniques as fluorescence and immunomagnetic may cause a serious threat to the life of the cells due to various compatibility issues. Moreover, they also require an extra pre-processing labeling step. Contrarily, label-free separation can be considered as an alternative solution to the traditional bio-particle separation methods, due to their simpler operating principles and lower cost constraints. Acoustic based particle separation methods have captured a lot of attention among the other reported label-free particle separation techniques because of the numerous advantages it offers. Practical implications This study tries to briefly cover the developments of different acoustic-based particle separation techniques over the years. Unlike the conventional surveys on general bioparticles separation, this study is focused particularly on the acoustic-based particle separation. The study would provide a comprehensive guide for the future researchers especially working in the field of the acoustics, in studying and designing the acoustic-based particle separation techniques. Originality/value The study insights a brief theory of different types of acoustic waves and their interaction with the bioparticles is considered, followed by acoustic-based particle separation devices reported till the date. The integration of acoustic-based separation techniques with other methods and with each other is also discussed. Finally, all major aspects like the approach, and productivity, etc., of the adopted acoustic particle separation methods are sketched in this article.

Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly

Cathepsin G (CatG) is responsible for several distinct immune processes of adaptive and innate immunity depending on extra- or intracellular occurrence of CatG. Recently, we established a method to detect CatG activity at the cell surface of natural killer cells by using the activity-based probe MARS116-Bt in flow cytometry. MARS116-Bt consists of biotin, spacer, amino acid sequence, and a phosphonate warhead which binds covalently to the serine amino acid residue within the active center of CatG. Herein, MARS116 was conjugated to 5(6)-carboxyfluorescein (FAM) in order to limit non-specific signal-to-noise ratio generally resulting from binding of fluorescein-labelled avidin (avidin-FAM) to biotinylated, intracellular proteins; since MARS116-Bt is incubated with avidin-FAM in a second labelling step. MARS116-FAM was capable to detect intracellular CatG activity, in contrast to the control compound MARS116*-FAM which lacks the functional phosphonate warhead crucial for binding to the active-site of CatG and contains a carboxyl group instead. Furthermore, intracellular CatG activity was determined in CD4+ T cells, CD8+ T cells as well as in T regulatory cell (Treg) subsets. Thus, MARS116-FAM is a convenient activity-based probe to detect intracellular CatG activity in a flow cytometry approach.

Graphene biosensor as affinity biosensors for biorecognition between Guanine riboswitch and ligand

Guanine riboswitch (GR) is located in the untranslated region of mRNA that performs important biological functions by interactions with specific ligand molecules. The present standard methods for detecting mRNA has been widely used, but suffer from extra labeling steps, high cost, low throughput, low sensitivities and limited dynamic ranges. The field effect transistor (FET) biosensors showed high performance in detecting a large number of biological analytes. However, the detection of analytes with low charge and small molecule still remains a challenge. In this paper, a label-free graphene field effect transistor (G-FET) biosensor has been developed to detect the binding interactions between GR and four purine analog ligand molecules (GUA, 6GU, 2BP, XAN). We demonstrated that the G-FET biosensor were highly sensitive in detecting the electric-neutral and low molecular weight ligands by using GR as probe. The sensing mechanisms for electric-neutral molecule is revealed by molecular dynamics simulation. The equilibrium dissociation constants KD and binding free energy ΔG of GR-GUA, GR-6GU, GR-2BP and GR-XAN were obtained by the G-FET biosensor, and the results are comparable with the calculated by molecular dynamics simulation and previous reports. We demonstrated that the G-FET biosensor can be used as affinity biosensors to quantify biomolecular interactions.

Learning a representation of tongue dynamics from unlabeled ultrasound videos

Ultrasound imaging of the tongue has been used for decades in studies of speech production and speech motor control, for silent speech interfaces, and in numerous other areas. Despite substantial efforts, however, extraction of reliable features from ultrasound tongue data remains a challenge due to speckle noise and acoustic propagation issues. Recently, Representation Learning has emerged in a variety of fields as a powerful means of generating useful representations of underlying structure in raw, high-dimensional data. In its unsupervised form, Representation Learning discovers structures in unlabelled data, thereby eliminating the need for a time-consuming labelling step. The present work is believed to be the first use of unsupervised Representation Learning to reveal structures related to tongue dynamics in unlabelled ultrasound video. A 3-D Convolutional Neural Network examining a series of unlabelled 60 Hz tongue images is found to accurately predict unseen future images even for large interframe tongue displacements. By comparing the 3DCNN prediction error to that of a simple previous-frame predictor, tongue trajectories containing transitions between regions of acoustic stability can be identified and correlated with formant trajectories in a spectrogram. Prospects for leveraging the tongue dynamic representation for use in subsequent speech processing tasks will be discussed.

Label-free G-Quadruplex aptamer and Thioflavin-T based turn-off fluorescent detection of ethanolamine

A label free aptasensor based on G-quadruplex forming ethanolamine aptamers and Thioflavin T was developed for the selective and sensitive detection of ethanolamine. The detection system consists of DNA aptamers with four GGG repeats forming a G-quadruplex that Thioflavin T can easily bind to causing high fluorescence emission intensity because of its fluorescence molecular rotor nature. In the presence of ethanolamine, binding of aptamer to its target induces conformational changes causing the separation of Thioflavin T from the complex with a dramatic decrease in fluorescence intensity of reaction mixture. This fluorescence aptasensor has a convenient sensitivity and selectivity with a detection limit of 641 μM to measure ethanolamine directly in contaminated water samples in a wide linear range. Furthermore this method avoids all complicated modification and labelling steps and thus offers a simple, fast, and cost efficient solution for ethanolamine detection.

Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry.

The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online.