Direct Labeling Research Articles

Coffee Roasters’ Sustainable Sourcing Decisions and Use of the Direct Trade Label

This paper analyzes motivations for coffee roasters to source directly from farmers and how roasters decide whether to use the Direct Trade sustainability label. Direct Trade is an uncertified label connoting an approach wherein roasters negotiate coffee price and quality with farmers without intermediaries, with purported farmer income benefits. We examine semi-structured interviews with 11 US roasters and three coffee stakeholders to identify motivations to source directly, provide customers sourcing information, and use or reject the Direct Trade label. We find that roasters directly source coffee primarily for quality reasons and communicate about sourcing because they believe customers would value coffee more if they understood their sustainable sourcing practices. However, the lack of a clear definition for the Direct Trade label, coffee roaster concerns about the label’s utility, and the threat of “free riders” disincentivizes label use. Without a shared label, customers face high costs for information about directly sourced coffee, which may limit the expansion of a sourcing practice that could benefit farmers.

A ribose-functionalized NAD+ with unexpected high activity and selectivity for protein poly-ADP-ribosylation

Nicotinamide adenine dinucleotide (NAD+)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD+ analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3′-OH of nicotinamide riboside enables enzymatic synthesis of an NAD+ analogue with high efficiency and yields. Notably, the generated 3′-azido NAD+ exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3′-azido NAD+ in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3′-azido NAD+ provides an important tool for studying cellular PARylation.

Unequivocal imaging of aluminium in human cells and tissues by an improved method using morin

Aluminium is biologically reactive and its ability to potentiate the immune response has driven its inclusion in both veterinary and human vaccines. Consequently, the need for unequivocal visualisation of aluminium in vivo has created a focused research effort to establish fluorescent molecular probes for this purpose. The most commonly used direct fluorescent labels for the detection of aluminium are morin (2′,3,4′,5,7-pentahydroxyflavone) and lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulphonic acid]. While the former has gained popularity in the detection of aluminium in plants and predominantly within root tips, the latter boasts greater sensitivity and selectivity for the detection of aluminium in human cells and tissues. Herein, we have developed a simplified morin staining protocol using the autofluorescence quenching agent, Sudan Black B. This modified protocol improves tissue morphology and increases analytical sensitivity, which allows intracellular aluminium to be detected in monocytes and when co-localised with senile plaques in human brain tissue of donors diagnosed with familial Alzheimer’s disease. Overall, our results demonstrate a simple approach to minimise false positives in the use of morin to unequivocally detect aluminium in vivo.

MRI Atlas of the Human Deep Brain.

Mastering detailed anatomy of the human deep brain in clinical neurosciences is challenging. Although numerous pioneering works have gathered a large dataset of structural and topographic information, it is still difficult to transfer this knowledge into practice, even with advanced magnetic resonance imaging techniques. Thus, classical histological atlases continue to be used to identify structures for stereotactic targeting in functional neurosurgery. Physicians mainly use these atlases as a template co-registered with the patient's brain. However, it is possible to directly identify stereotactic targets on MRI scans, enabling personalized targeting. In order to help clinicians directly identify deep brain structures relevant to present and future medical applications, we built a volumetric MRI atlas of the deep brain (MDBA) on a large scale (infra millimetric). Twelve hypothalamic, 39 subthalamic, 36 telencephalic, and 32 thalamic structures were identified, contoured, and labeled. Nineteen coronal, 18 axial, and 15 sagittal MRI plates were created. Although primarily designed for direct labeling, the anatomic space was also subdivided in twelfths of AC-PC distance, leading to proportional scaling in the coronal, axial, and sagittal planes. This extensive work is now available to clinicians and neuroscientists, offering another representation of the human deep brain ([https://hal.archives-ouvertes.fr/] [hal-02116633]). The atlas may also be used by computer scientists who are interested in deciphering the topography of this complex region.

Indirect Labeling of DNA for Microarrays.

As with RNA labeling protocols, the main difference between direct and indirect DNA labeling protocols is a trade-off of cost and time. Indirect labeling of DNA, described here, takes ∼2 h longer than direct labeling but is hundreds of dollars cheaper.

Direct Cyanine-dCTP Labeling of DNA for Microarrays.

Similar to direct RNA labeling, direct DNA labeling with Cy-dCTP is the simplest and fastest method for labeling DNA. This is a standard Klenow labeling protocol in which Cy-dCTP is incorporated during the labeling reaction. After stopping the reaction, labeled nucleotide is separated from unreacted Cy-dCTP, and Cy3- and Cy5-labeled materials are combined for hybridization. This protocol is suitable for many applications, including detection of copy-number variation, nucleosome mapping, and other location analysis (e.g., chromatin immunoprecipitation [ChIP]-chip).

Direct fluorescent glycan labeling with recombinant sialyltransferases.

Glycosylation is a common modification found on numerous proteins and lipids. However, direct detection of glycans on these intact biomolecules has been challenge. Here, utilizing enzymatic incorporation of fluorophore-conjugated sialic acids, dubbed as direct fluorescent glycan labeling, we report the labeling and detection of N- and O-glycans on glycoproteins. The method allows detection of specific glycans without the laborious gel blotting and chemiluminescence reactions used in Western blotting. The method can also be used with a variety of fluorescent dyes.

Specific Targeting of Somatostatin Receptor Subtype-2 for Fluorescence-Guided Surgery

Clinically available intraoperative imaging tools to assist surgeons in identifying occult lesions are limited and partially responsible for the high rate of disease recurrence in patients with neuroendocrine tumors (NET). Using the established clinical efficacy of radiolabeled somatostatin analogs as a model, we demonstrate the ability of a fluorescent somatostatin analog to selectively target tumors that overexpress somatostatin receptor subtype-2 (SSTR2) and demonstrate utility for fluorescence-guided surgery (FGS). A multimodality chelator (MMC) was used as a "radioactive linker" to synthesize the fluorescently labeled somatostatin analog, 67/68Ga-MMC(IR800)-TOC. In vivo studies were performed to determine the pharmacokinetic profile, optimal imaging time point, and specificity for SSTR2-expressing tissues. Meso- and microscopic imaging of resected tissues and frozen sections were also performed to further assess specific binding, and binding to human NETs was examined using surgical biospecimens from patients with pancreatic NETs. Direct labeling with 67Ga/68Ga provided quantitative biodistribution analysis that was in agreement with fluorescence data. Receptor-mediated uptake was observed in vivo and ex vivo at the macro-, meso-, and microscopic scales. Surgical biospecimens from patients with pancreatic NETs also displayed receptor-specific agent binding, allowing clear delineation of tumor boundaries that matched pathology findings. The radioactive utility of the MMC allowed us to validate the binding properties of a novel FGS agent that could have a broad impact on cancer outcomes by equipping surgeons with real-time intraoperative imaging capabilities.

Antibody-Free Assay for RNA Methyltransferase Activity Analysis.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

68Ga-Glutathione radio-complex as a new diagnostic probe for targeting colon cancer in rats.

Glutathione (GSH) plays an important role in a horde of cellular events that include cell proliferation and apoptosis.The present study describes the radio-synthesis and characterization of gallium-68 (68Ga)-labelled glutathione for its application in radionuclide imaging of cancer. The radio-synthesis of radio-complex 68Ga-GSH was performed by the direct labeling method. The developed radio-complex was subjected to quality control tests. Colon tumors were developed in healthy male Sprague Dawley (S.D) rats by giving subcutaneous injections of dimethylhydrazine (DMH) in order to monitor the uptake of 68Ga-GSH radio-complex. Gallium-68-labelled glutathione was synthesized with a labeling efficiency of 73.5%±1%. Percentage plasma protein binding and log Po/w values for the radio-complex were found to be 20%-30% and -0.223±0.12, respectively. A significantly higher percentage specific uptake value of newly developed 68Ga-GSH complex was observed in colon tumor in comparison to soft tissue at 90 minutes post administration thereby exhibiting specificity for cancerous cells, which was also witnessed significantly increased overtime from the ratio of colon tumor uptake to normal colon uptake (P≤0.05). Therefore, 68Ga-labelled glutathione can further be exploited for radionuclide imaging and assessment of tumor drug resistance in patients.

Abstract 5125: A novel method for isolating high-quality UHMW DNA from 10 mg of freshly frozen or liquid-preserved animal and human tissue including solid tumors

Abstract High-quality UHMW DNA is key to success in optical mapping of long genomic DNA using Bionano Genomics Saphyr® system for de novo genome assembly and structural variation detection. Ideally, ~100 mg of tissue is frozen immediately upon collection using liquid nitrogen or dry ice, and stored at -80°C before DNA isolation. Practically, tissue amounts may be limited and collection often occurs at sites where low temperature preservation for storage and shipping may be unavailable. Here we present a novel method to isolate UHMW DNA from 10 mg of either freshly frozen or room temperature preserved animal and human tissue for subsequent enzymatic labeling using direct labeling and staining (DLS) to generate Bionano maps. With a TissueRuptor, 10 mg tissue is homogenized followed by a three-step purification process before embedding the purified nuclei in agarose gel plugs. This process takes less than 3 hours for a batch of 6 samples. Using this method, we have successfully isolated high quality UHMW DNA from various tissue types of rat and human including solid tumors. The method is very attractive compared to other protocols because: 1) tissue preservation, shipping and handling can be done at room temperature, 2) uses very small amount of tissue - possible application for rare samples and human tissue biopsy tests, and 3) the length and quality of the resulting single molecules generate high quality optical maps for genome finishing and SV calling. Citation Format: Yang Zhang, James Broach. A novel method for isolating high-quality UHMW DNA from 10 mg of freshly frozen or liquid-preserved animal and human tissue including solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5125.

Abstract 1133: ImmunoPET and T cell labeling for in vivo monitoring of immunotherapy: Development of CD8 Fab2’ and Fab tracers for immunoPET and direct labeling of T cells for in vivo tracking

Abstract Objectives: Cancer immunotherapy is becoming an increasingly important treatment strategy across a broad spectrum of tumor types. Non-invasive imaging methods for in vivo tracking of T-cells can be used for longitudinal monitoring of tumor infiltrating T-cells to assess the effect of immune checkpoint therapy or cell-based immunotherapies. The objective of the current work was to develop and optimize Fab-based tracers directed at CD8 for PET imaging of T cells. The imaging performance of radiolabeled Fab fragments was compared to Fab2’ fragments in terms of kinetics and image contrast. Furthermore, Zirconium-89 (89Zr) labeled T cells were tracked in a murine transplantation model. Methods: Anti-mouse CD8α antibody was cleaved into Fab2’ by enzymatic cleavage followed by reduction of disulfide bridge into Fab fragments. Fab2’ was conjugated to NCS-DFO for 89Zr labeling, while both Fab2’ and Fab were conjugated to SCN-NOTA for Cupper-64 (Cu-64) labeling. Radiochemical yield, radiochemical purity (RCP) and plasma/buffer stability was assessed by high-performance liquid chromatography. Furthermore, the immunoreactivity and binding affinity was evaluated for the radiolabeled tracers. In vivo comparison of radiolabeled Fab2’ and Fab fragments was performed by longitudinal PET/CT imaging at various time-points after injection in CT26 (murine colon carcinoma) tumor bearing mice. CD3+ T-cells were directly labeled with 89Zr for tracking experiments. The labeling efficiency and viability of the cells was evaluated ex vivo, and in vivo cell tracking was performed by longitudinal PET/CT imaging after injection of labeled T-cells in naive immune-competent mice. Results: The cleavage of anti-CD8 into Fab2’ and subsequent purification by HPLC was optimized to achieve a product with high yield and purity. The optimal Fab2’ reduction conditions were determined followed by conjugation to SCN-NOTA An optimized protocol for 89Zr/64Cu-CD8-Fab2’ and 64Cu-CD8-Fab was developed with high radiochemical yield and RCP of >99%. PET/CT imaging with all tracers was performed to compare tumor, spleen and lymph node uptake. The highest uptake was found 24h post injection, but a clear delineation of lymphoid organs and tumors was evident already after 6h for the single Fab allowing same day imaging. T-cells labeled with 89Zr and cell tracking PET/CT imaging showed homing primarily to the spleen. Conclusions: Different tracers directed at T-cells were successfully developed and evaluated in in vivo PET imaging of T cells. The reduced circulation time of Fab vs. Fab2’ and the use of 64Cu instead of 89Zr allows repeated imaging every 3 days due to the shorter physical half-life (12.7 h) of 64Cu. The study furthermore demonstrates the use of direct radiolabeling of T-cells as an attractive tool for investigating the distribution of cell-based therapies. Citation Format: Camilla Christensen, Lotte K. Kristensen, Carsten H. Nielsen, Andreas Kjær. ImmunoPET and T cell labeling for in vivo monitoring of immunotherapy: Development of CD8 Fab2’ and Fab tracers for immunoPET and direct labeling of T cells for in vivo tracking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1133.

Direct Cyanine-dUTP Labeling of RNA for Microarrays.

This is the simplest method to label RNA for use in expression analysis. RNA is reverse-transcribed using both oligo(dT) and random hexamers as primers. The random hexamers improve overall efficiency of labeling, especially at the 5' end of the RNA. Fluorescently labeled dUTP is incorporated into the cDNA. After reverse transcription, the RNA is degraded, and the labeled cDNA is purified from unincorporated Cy dyes. Finally, samples labeled with Cy3 and Cy5 dyes are mixed and combined with blocking nucleotides and used for hybridization.

Indirect Radioiodination of DARPin G3 Using N-succinimidyl-Para-Iodobenzoate Improves the Contrast of HER2 Molecular Imaging.

Radionuclide molecular imaging of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal cancer might be used to stratify patients for HER2-targeted therapy as well as monitor treatment response and disease progression. Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins with favorable properties for molecular imaging. Herein we compared two methods for labeling the anti-HER2 DARPin (HE)3-G3, direct and indirect radioiodination. We hypothesized that the use of N-succinimidyl-para-iodobenzoate (SPIB) for radioiodination would facilitate the clearance of radiometabolites and improve the contrast of imaging. Both radiolabeled (HE)3-G3 variants preserved their binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was also demonstrated. A biodistribution comparison of [125I]I-(HE)3-G3 and [125I]I-PIB-(HE)3-G3, in mice bearing HER2 expressing SKOV3 xenografts, showed rapid clearance of [125I]I-PIB-(HE)3-G3 from normal organs and tissues and low accumulation of activity in organs with NaI-symporter expression. Both radiolabeled (HE)3-G3 variants had equal tumor uptake. Consequently, the indirect label provided higher tumor-to-blood and tumor-to-organ ratios compared with the direct label. Comparative Single Photon Emission Computed Tomography (SPECT)/CT imaging of HER2 expression in SKOV3 xenografts, using both radiolabeled DARPins, demonstrated the superior imaging contrast of the indirect label. Indirect radioiodination of (HE)3-G3 using SPIB could be further applied for SPECT and PET imaging with iodine-123 and iodine-124.

Atlas of Subcellular RNA Localization Revealed by APEX-Seq

We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq shouldbe widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

Synthesis and preliminary in vitro evaluation of DOTA-Tenatumomab conjugates for theranostic applications in tenascin expressing tumors

Tenatumomab is an anti-tenascin murine monoclonal antibody previously used in clinical trials for delivering radionuclides to tumors by both pre-targeting (biotinylated Tenatumomab within PAGRIT) and direct 131Iodine labeling approaches. Here we present the synthesis and in vitro characterization of three Tenatumomab conjugates to bifunctional chelating agents (NHS-DOTA, NCS-DOTA and NCS-DTPA). Results indicate ST8198AA1 (Tenatumomab-DOTAMA, derived by conjugation of NHS-DOTA), as the most promising candidate in terms of conjugation rate and yield, stability, antigen immunoreactivity and affinity. Labeling efficiency of the different chelators was investigated with a panel of cold metals indicating DOTAMA as the best chelator. Labeling of Tenatumomab-DOTAMA was then optimized with several metals and stability performed confirms suitability of this conjugate for further development. ST8198AA1 represents an improvement of the previous antibody forms because the labeling with radionuclides like 177Lu or 64Cu would allow theranostic applications in patients bearing tenascin expressing tumors.

Pre-labelling versus direct labelling of anthrax proteins for imaging of matrix metalloproteinases activity using DOTA-GA

IntroductionIncreased activity of matrix metalloproteases (MMPs) is associated with reduced survival in several cancer subtypes. Aiming to produce an MMP tumour cell-selective cytotoxin, we genetically modified both components of the AB–type lethal toxin from Bacillus anthracis. Component A, Protective Antigen (PA-WT), was re-engineered to form an oligomeric pore in cell membranes only when cleaved by MMPs (PA-L1). The pore-translocation domain (LFn – N-terminal, 30 kDa) of the Lethal Factor (LF), component B, was fused to the catalytic domain of Pseudomonas exotoxin-A to increase its cytotoxic effect when delivered to cancerous cells. Here, we develop radiolabelled forms of LFn for MMP activity imaging by SPECT using the LFn/PA-L1 system. MethodsDOTA-GA-maleimide was conjugated to LFn to allow radiolabelling with 111In via two different routes: (1) LFn was conjugated with maleimide-DOTA-GA under mild conditions, and then radiolabelled in acidic conditions at 95°C, or (2) 111In was coordinated to maleimide-DOTA-GA first and then conjugated via maleimide chemistry to LFn. Circular Dichroism Spectroscopy of LFn was performed to evaluate changes in its secondary structure. Cell uptake assays using the differently labelled forms of [111In]In-DOTA-GA-LFn in the presence or not of PA-WT or PA-L1 were performed. ResultsLFn was successfully radiolabelled by either strategy. Comparison of the secondary structure content of LFn exposed to 37°C or 95°C, showed a loss of alpha helix content at higher temperatures. Cell uptake of both forms of [111In]In-DOTA-GA-LFn, labelled directly or indirectly, was significantly higher in MMP-positive cells, in the presence of PA-L1, compared to controls. Notably, despite being exposed to high temperatures, uptake of directly labelled [111In]In-DOTA-GA-LFndir was higher than indirectly labelled [111In]In-DOTA-GA-LFnindir. Conclusions111In-radiolabelling of LFn results in a functional molecule that targets MMP-activity in cells when combined with PA-L1. [111In]In-LFn/PA-L1 is a promising MMP activity imaging agent for SPECT imaging.

SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays.

This study presents a novel size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method for the characterisation and quantification of immunoassays with lanthanide-labelled antibodies. SEC-ICP-MS in combination with a double isotope dilution approach enabled facile validation of the antibodies' integrity, the determination of the batch to batch labelling efficiency, monitoring of each labelling step, and quantification of the immunocomplexes after incubation with the target protein. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD = 3.7ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid chromatographic method with separation times under 7min of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided valuable insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an 153Eu-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with 156Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing 153Eu-labelled polyclonal antibodies. Graphical abstract.

Directed Iridium-Catalyzed Hydrogen Isotope Exchange Reactions of Phenylacetic Acid Esters and Amides.

For the first time, a catalytic protocol for a highly selective hydrogen isotope exchange (HIE) of phenylacetic acid esters and amides under very mild reaction conditions is reported. Using a homogeneous iridium catalyst supported by a bidentate phosphine-imidazolin-2-imine P,N ligand, the HIE reaction on a series of phenylacetic acid derivatives proceeds with high yields, high selectivity, and with deuterium incorporation up to 99 %. The method is fully adaptable to the specific requirements of tritium chemistry, and its effectiveness was demonstrated by direct tritium labeling of the fungicide benalaxyl and the drug camylofine. Further insights into the mechanism of the HIE reaction with catalyst 1 have been provided utilizing DFT calculations, NMR studies, and X-ray diffraction analysis.

Designed Synthesis of Dual Emitting Silicon Quantum Dot for Cell Imaging: Direct Labeling of Alpha 2-HS-Glycoprotein.

The innocent silicon quantum dots (SQDs) having dual emissive property (blue in VIS and red in NIR), high photostability, and freedom from auto fluorescence are designed and synthesized for the first time using ethylene glycol. A new attempt has been made for direct labeling of Alpha 2-HS-Glycoprotein (Fetuin A) through functionalization of the synthesized dots by EDC coupling. The SQDs were characterized by FTIR, TEM, AFM, XRD, EDX, DLS, and TGA. The chemistry involved in the synthesis and functionalization of dots is elucidated in detail. The synthesized SQDs are suitable for live cell imaging as well as direct labeling of the Fetuin A in the NIR region. The direct labeling technique developed for Fetuin A imaging is robust, more specific, and simple, and reduces the number of incubation and washing steps and produces better quality data compared to the conventional method using Rhodamine B.