Smart Pipette: Elevating Laboratory Performance with Tactile Authenticity and Real-Time Feedback.

Mastering the correct use of laboratory equipment is a fundamental skill for undergraduate science students involved in laboratory-based training. However, hands-on laboratory time is often limited, and remote students may struggle as their absence from the physical lab limits their skill development. An air-displacement micropipette was selected for our initial investigation, as accuracy and correct technique are essential in generating reliable assay data. Handling small liquid volumes demands hand dexterity and practice to achieve proficiency. This research assesses the importance of tactile authenticity during training by faithfully replicating the micropipette's key physical and operational characteristics. We developed a custom haptic training approach called 'Smart Pipette' which promotes accurate operation and enhances laboratory dexterity training. A comparative user study with 34 participants evaluated the effectiveness of the Smart Pipette custom haptic device against training with off-the-shelf hardware, specifically the Quest VR hand controller, which was chosen because it is held mid-air similar to a laboratory micropipette. Both training conditions are integrated with the same self-paced virtual simulation displayed on a computer screen, offering clear video instructions and realtime guidance. Results demonstrated that participants trained with the Smart Pipette custom haptic exhibited increased accuracy and precision while making fewer errors than those trained with off-the-shelf hardware. The Smart Pipette and the Quest VR controller had no significant differences in cognitive load and system usability scores. Tactile authentic interaction devices address challenges faced by online learners, while their applicability extends to traditional classrooms, where real-time feedback significantly enhances overall training performance outcomes.

Interdisciplinary teaching squares enhance reflection and collegiality: A collaborative pedagogical approach.

AIM: To explore the experiences of interdisciplinary Higher Educational Academics within Nursing, Midwifery, Pharmacy and Biomedical Science in the use of teaching squares as a formative, collaborative pedagogical tool to promote reflection. BACKGROUND: One approach to enhancing academic teaching practice involves the provision of feedback to individual academics. This approach can be challenging for the reviewer, hence other less intimidating approaches are popular. One such approach is the teaching square. In this approach typically 4 other teaching staff are involved in observing their peers' teaching methods (synchronous or asynchronous) and then engaging in a cycle of personal reflection. Reflection on teaching practices of their peers can provide opportunity to improve their own teaching. Typically teaching squares involve participants from the same academic discipline, however, in this study participants from related yet different disciplines were purposely connected and the benefits of this approach assessed. DESIGN: A qualitative descriptive design was used to explore the participants' experiences of undertaking interdisciplinary teaching squares through online questionnaires and focus group feedback opportunities. METHODS: This study was undertaken between August 2022 to June 2023 at an Australian university. Sixteen academic staff members from Nursing, Midwifery, Pharmacy & Pharmaceutical science and Biological science expressed an interest in the project. Five participants elected not to continue citing time pressures and 11 staff members participated in the project. Participation involved completing the teaching square experience and a subsequent focus group that were held to explore their experiences of undertaking a teaching square. The demographic survey data were presented and the focus group interviews were recorded, transcribed and analysed thematically. RESULTS: Triangulation of the findings resulted in the identification of four main themes: Teaching squares for professional networks; Perceptions of Safety; Stepping in and out of a reflective cycle; and Time Constraints vs. Time Value. CONCLUSIONS: The study aimed to explore the effectiveness of teaching squares in encouraging academic reflection on teaching and fostering a collaborative teaching culture within interdisciplinary higher education academics. An unexpected finding was the value and promotion of interdisciplinarity professional relationships and networks. The findings from this research project offer valuable insights into the benefits of adopting teaching squares in health education and contributes to evidence-based pedagogical practices.

Interdisciplinary reflection by higher education academics using teaching squares: A scoping review.

OBJECTIVE: To explore the use of teaching squares by interdisciplinary Higher Education (HE) academics when engaging in a cycle of teaching reflection. DESIGN: A scoping review of published and unpublished research between 2012 and 2022. DATA SOURCES: Systematic search of ten (10) electronic databases and hand searching of reference lists identified 13 studies for review. REVIEW METHODS: Studies were included if reflection was undertaken on teaching and involved the disciplines of Nursing, Midwifery, Pharmacy, and Biomedical Sciences. The data were extracted and charted and presented using the Patterns, Advances, Gap, Evidence for practice and Research [PAGER] framework. RESULTS: The main theme identified in the review was that teaching squares led to the development of improved pedagogical skills. This skills improvement was facilitated by the creation of positive academic relationships formed by undertaking interdisciplinary observation, reflection and other serendipitous events. HE academics gained positive benefits from this process, especially those newly transitioning into academia. Some examples of these benefits included increased awareness of one's own teaching practice, deeper understanding of the student experiences and the HE academic feeling less isolated and more reassured about their teaching. Undertaking interdisciplinary reflection led to the development of social capital, resulting in increased confidence. This was evident by the development of new professional relationships from increased networking opportunities external to the faculty in which the HE academic was located. The culture within each context served as either a barrier or facilitator to engaging in reflection. We also noted there were a variety of ways in which reflection was being undertaken, with new insights gained during COVID-19. CONCLUSION: This scoping review explored the current published literature on reflection on teaching undertaken by HE academics within Nursing, Midwifery, Pharmacy and Biomedical Science disciplines. The key outcomes for the interdisciplinary stakeholders were increased levels of confidence, learning of new ways of teaching, and insight into the student experience by undertaking interdisciplinary reflection. From a faculty perspective this is meant there was an increase of social network development and provided higher levels of social capital, especially for those transitioning into academia. The pandemic led to an increased reliance on reflection of virtual reflection, which may become the norm. Further research is required to explore the experiences and perceptions of reflection for this cohort of HE teachers.

A combined interactive online simulation and face-to-face laboratory enable undergraduate student proficiency in hemocytometer use, cell density and viability calculations.

A hemocytometer is a key piece of laboratory equipment typically used in diagnostic and immunology research laboratories to enumerate white blood cells. The accurate quantification of cell density is essential to ensure accurate numbers of cells are added to assays to generate valid data. Hence, learning to correctly use a hemocytometer is a critical skill for all undergraduate immunology students. However, this skill can be challenging to learn because of students' unfamiliarity with correct cell identification, differentiating viable versus dead cells and mathematical proficiency in calculating cell density and viability. To address these issues, we developed an interactive computer simulation that replicated all aspects of a Neubauer-style hemocytometer. This simulation was used to teach second-year undergraduate immunology students before a face-to-face (F-2-F) laboratory exercise where these skills were applied. Using a mixed methods approach, student performance and feedback were collected on broad aspects of the intervention and its benefits to the F-2-F setting. The approach was found to be extremely successful with all measures indicating a significant impact of the virtual hemocytometer on student learning, understanding and confidence. We suggest that integrating an online simulation to teach students the fundamentals of hemocytometer use and calculations is a valuable educational aid for learning this important skill.

Enhancing teaching effectiveness in biochemistry labs: Author reflections and improvement strategies.

This article details the outcome of a joint reflective approach undertaken by the authors to identify common difficulties experienced by 2nd-year undergraduate Biochemistry students in laboratory classes. Difficulties experienced in laboratories can affect the development of hand skills, an understanding of how to correctly operate laboratory equipment and the linkage between didactic content and their experimental demonstration. These difficulties covered were identified based on their common appearance across multiple cohorts and are grouped into five broad areas. The context of the laboratory exercises is detailed and the common difficulties experienced by students are outlined. The potential causes of these difficulties are then discussed along with the approaches and strategies that were implemented to help resolve future occurrences. The approach and resources developed to address these difficulties may help other Biochemistry educators who are facing similar experiences with their undergraduate students.

Intracellular immunoglobulin A (icIgA) in protective immunity and vaccines.

Virus neutralization at respiratory mucosal surfaces is important in the prevention of infection. Mucosal immunity is mediated mainly by extracellular secretory immunoglobulin A (sIgA) and its role has been well studied. However, the protective role of intracellular specific IgA (icIgA) is less well defined. Initially, in vitro studies using epithelial cell lines with surface expressed polymeric immunoglobulin receptor (pIgR) in transwell culture chambers have shown that icIgA can neutralize influenza, parainfluenza, HIV, rotavirus and measles viruses. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across the polarized cell. Co-localization of specific icIgA with influenza virus in patients' (virus culture positive) respiratory epithelial cells using well-characterized antisera was initially reported in 2018. This review provides a summary of in vitro studies with icIgA on colocalization and neutralization of the above five viruses. Two other highly significant respiratory infectious agents with severe global impacts viz. SARS-2 virus (CoViD pandemic) and the intracellular bacterium-Mycobacterium tuberculosis-are discussed. Further studies will provide more detailed understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps with a specific focus on mucosal infections. This will inform the design of more effective vaccines against infectious agents transmitted via the mucosal route.

From the bench to the lectern: teaching immunology in higher education as a rewarding career opportunity.

This article covers the career pathway of a research-trained scientist to an immunology educator in a university setting.

Introducing First-Year Undergraduate Students the Fundamentals of Antibiotic Sensitivity Testing through a Combined Computer Simulation and Face-to-Face Laboratory Session.

Determining the antibiotic sensitivity of disease-causing microorganisms is a fundamental process in a clinical microbiology laboratory. With the continued use of antibiotics, the emergence of antibiotic resistance has become a significant health issue. However, the principles and laboratory testing to determine antibiotic sensitivity are generally not taught to first-year undergraduate students. This is partly due to the limited time to cover the fundamental biology of microorganisms and the mechanism of action of antibiotics in an introductory course. We overcame these limitations by teaching first-year students the fundamental principles of antibiotic sensitivity using an online data generator/simulation. Using the Kirby-Bauer disk diffusion test, students replicated the effects of antibiotic dose on bacterial growth and determined the antimicrobial susceptibility testing of their allocated bacterium. After 2-3 weeks, the antimicrobial sensitivity testing was replicated in an authentic face-to-face laboratory setting over 2 days. The impact of the intervention on student learning was assessed using a written laboratory report and a short questionnaire containing Likert and free-text questions. Student self-reported understanding of the content rose significantly, with nearly all students passing the written assessment. The approach was found to be enjoyable and interactive and facilitated authentic learning in first-year students. This cohort of students will continue to use more advanced versions of this simulation in future years, allowing for the long-term benefits of this approach to be assessed.

Methylglyoxal Impairs Sister Chromatid Separation in Lymphocytes.

The accurate segregation of sister chromatids is complex, and errors that arise throughout this process can drive chromosomal instability and tumorigenesis. We recently showed that methylglyoxal (MGO), a glycolytic by-product, can cause chromosome missegregation events in lymphocytes. However, the underlying mechanisms of this were not explored. Therefore, in this study, we utilised shotgun proteomics to identify MGO-modified proteins, and label-free quantitation to measure changes in protein abundance following exposure to MGO. We identified numerous mitotic proteins that were modified by MGO, including those involved in the separation and cohesion of sister chromatids. Furthermore, the protein abundance of Securin, an inhibitor of sister chromatid separation, was increased following treatment with MGO. Cytological examination of chromosome spreads showed MGO prevented sister chromatid separation, which was associated with the formation of complex nuclear anomalies. Therefore, results from this study suggest MGO may drive chromosomal instability by preventing sister chromatid separation.

Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress.

Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all samples. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Folic acid deficiency increases sensitivity to DNA damage by glucose and methylglyoxal.

Type 2 diabetes (T2D) is associated with elevated frequencies of micronuclei (MNi) and other DNA damage biomarkers. Interestingly, individuals with T2D are more likely to be deficient in micronutrients (folic acid, pyridoxal-phosphate, cobalamin) that play key roles in one-carbon metabolism and maintaining genomic integrity. Furthermore, it has recently been shown that deficiencies in these nutrients, in particular folic acid leaves cells susceptible to glucose-induced DNA damage. Therefore, we sought to investigate if the B lymphoblastoid WIL2-NS cell line cultured under folic acid-deficient conditions was more sensitive to DNA damage induced by glucose, or the reactive glycolytic byproduct methylglyoxal (MGO) and subsequent advanced glycation endproduct formation. Here, we show that only WIL2-NS cultured under folic acid-deficient conditions (23 nmol/l) experience an increase in MNi frequency when exposed to high concentrations of glucose (45 mmol/l) or MGO (100 µmol/l). Furthermore, we showed aminoguanidine, a well-validated MGO and free radical scavenger was able to prevent further MNi formation in folic acid-deficient cells exposed to high glucose, which may be due to a reduction in MGO-induced oxidative stress. Interestingly, we also observed an increase in MGO and other dicarbonyl stress biomarkers in folic acid-deficient cells, irrespective of glucose concentrations. Overall, our evidence shows that folic acid-deficient WIL2-NS cells are more susceptible to glucose and/or MGO-induced MNi formation. These results suggest that individuals with T2D experiencing hyperglycemia and folic acid deficiency may be at higher risk of chromosomal instability.

Role of saturated and unsaturated fatty acids on dicarbonyl-albumin derived advanced glycation end products in vitro.

Glycation is a non-enzymatic reaction that occurs between the free amino group of proteins and reducing sugars and/or lipids, leading to the formation of advanced glycation end products (AGEs). The reaction also produces reactive oxygen species that have detrimental effects on cellular and extracellular proteins. Aminoguanidine is a known inhibitor of AGEs, and some fatty acids are known to have a beneficial role in vivo by reducing inflammation and oxidative stress. However, the role of fatty acids on AGE formation has not been thoroughly reported. We investigated the role of a range of fatty acids in the formation of AGEs and their reactive intermediates using an in vitro BSA-dicarbonyl model. The model assessed a time-dependent (0-72 h) and dicarbonyl concentration (0-2 mM) -dependent studies for the optimal formation of AGEs. A 72 h time point was found to be optimal for the reaction of BSA with either methylglyoxal (MGO) or glyoxal (GO) to generate AGE-BSA complexes. When arachidonic, eicosapentaenoic or docosahexaenoic acids were included in the reaction, a significant decrease in protein-bound fluorescent AGEs was seen compared to the respective controls. In contrast, saturated and 18 carbon polyunsaturated fatty acids showed no significant activity. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed saturated fatty acids significantly decreased the production of Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) from GO and MGO models, respectively, whilst increasing methylglyoxal-derived hydroimidazolone (MG-H1). In contrast, arachidonic, eicosapentaenoic and docosahexaenoic acids did not significantly change either CEL or MG-H1 compared to no treatment controls whilst significantly reducing CML levels.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

Design, implementation, and assessment of an interactive simulation to teach undergraduate immunology students hemolytic disease of the newborn.

Hemolytic disease of the newborn (HDN) is a potentially fatal condition caused by a Rhesus (Rh) antigen incompatibility between a mother and fetus. As a result, determining the Rh status of expectant parents is a routine clinical assessment. Both the physiological and immunological basis of this condition are taught to undergraduate students. At the University of South Australia, some undergraduate immunology students find this topic challenging. The author designed, implemented, and assessed the impact of an interactive simulation to facilitate student learning of HDN. The students were actively engaged in determining the blood grouping and Rh status of an expectant mother and father and then determining the possibility of developing HDN. The simulation was found to take only 15 min to complete yet led to a significant increase in student performance in an end of semester exam question. Student perceived understanding was found to significantly improve following the introduction of the simulation, even though the content had been covered in a formal lecture. Student feedback was highly positive of this learning approach. In conclusion, short, interactive simulations can be used effectively to enhance student learning of challenging concepts.

Using a science simulation-based learning tool to develop students' active learning, self-confidence and critical thinking in academic writing.

Simulation is one teaching strategy that health educators use to promote active learning, however, evidence to support whether students utilising simulations develop skills in critical thinking and appraisal in their academic literacy, is lacking. This study explored the impact of embedding a desktop simulation about the hemolytic disease of the newborn (HDN) in a first-year, undergraduate nursing course on students' acquisition of knowledge, self-confidence, and development of critical thinking in academic writing. The study used a quasi-experimental, two-group, and pre-test-post-test design. After using the simulation, students reported i. a positive learning experience, ii. an increase in their self-confidence to critically appraise a case study, and iii. greater student success in two assessments for student's participating in the simulation compared to students who only used lecture notes as a study tool. The findings support the use of simulations to promote active learning, knowledge acquisition and opportunities to develop self-confidence and critical thinking in academic writing.

Using online simulations to teach biochemistry laboratory content during COVID-19.

At the University of South Australia (UniSA), Biochemistry is a second year undergraduate course. The student cohort is diverse, with students enrolled in courses with a laboratory focus, such as Laboratory Medicine, Medical Science, Nutrition and Food Science and Pharmaceutical Science. The course is taught in a traditional manner, with weekly lectures, fortnightly tutorials and three practical sessions. In response to the growing numbers of COVID-19 cases, in mid-March the University leadership moved to cease face-to-face teaching. By this time, 58 of 96 students had completed the first two (of three) face-to-face laboratory practicals. In response to this decision, teaching of all practical based content was moved online for all students. The first question was, how do we teach practical content online? And secondly, how do we teach hands-on skills? The first question was addressed using a suite of online simulations, progressively developed since 2013. Simulations are widely used and shown to be useful as teaching aids in STEM. A total of five simulations were introduced each covering key aspects of laboratory practice, including fundamental mathematical skills, reading, and setting a pipette, basic Biochemistry assays, protein quantification, and enzyme kinetics. The second issue of teaching hands on skills was addressed once restrictions were eased. Students were invited to attend the laboratory to learn the kinesthetic skills with instructor guidance. Both approaches used proved to be highly effective and can be readily adapted not only to teaching Biochemistry, but any aspect of science education.

Manipulating leukocyte populations to mimic immune disease states: a novel active approach to teaching flow cytometry to undergraduate immunology students.

Flow cytometry detects and measures the physical and chemical characteristics of cells or particles. In medical laboratories, flow cytometers are used to quantify changes in cell populations associated with disease states, such as AIDS. While a powerful technique, it is challenging to teach the principles of flow cytometry to undergraduate students. One approach is to have students process and analyze a patient sample. However, this is not possible when the patient has an infectious disease. Here we report a two-stage approach to address this challenge. Magnetic beads were used to manipulate leukocytes cell populations in healthy blood to mimic the phenotype of eight immune disease conditions. The cells were then stained against cell surface markers for cell populations and analyzed by flow cytometry. The second stage focused on teaching flow cytometry over 2 wk. Week 1 involved a lecture, followed by a laboratory session where students learned how to stain a blood sample. In week 2, students worked in a computer pool to analyze the previously generated data and determine the immunological status of a control and patient sample. Using this approach, all students achieved 100% correct diagnosis of both control and patient samples. Student feedback via a questionnaire was overwhelmingly positive, and student perceived knowledge of flow cytometry increased after the session significantly. We effectively mimicked several disease states, eliminating the need to source patient samples, yet still teaching undergraduate students the principles of flow cytometry.

Kelch-like protein 5-mediated ubiquitination of lysine 183 promotes proteasomal degradation of sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.

Measuring the Asian seabass (Lates calcarifer) neutrophil respiratory burst activity by the dihydrorhodamine-123 reduction flow cytometry assay in whole blood.

The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.