MICy: a Novel Flow Cytometric Method for Rapid Determination of Minimal Inhibitory Concentration

MICy is a new, simple and rapid flow cytometry based antibiotic susceptibility testing (AST) method that produces susceptibility profile a workday earlier than the microdilution method or other classical phenotypic AST methods. Shortening the length of AST can accelerate clinical decision-making as targeted antibiotic treatment improves clinical outcomes and reduces mortality, duration of artificial ventilation, and length of stay in intensive care unit.

MiR-382-5p’s Function as a Suppressor in Osteosarcoma (OS) by Targeting PDPK1

ABSTRACT miR-382-5p engages in development of osteosarcoma (OS). However, the regulatory system of miR-382-5p in osteosarcoma remains to be revealed. This research studied the interplay between PDPK1 and miR- 382-5p in OS. RT-PCR was used to evaluate miR-382-5p and PDPK1 expression in OS cells and normal human osteoblast cells. Dual-luciferase reporter assay validated PDPK1 as a miR-382-5p target. CCK-8 evaluated the cell viability. Flow cytometric method determined cell apoptosis rate. Transwell and Scratch assays estimated the cell metastasis. miR-382-5p was inhibited in OS cells. Further functional results showed miR-382-5p upregulation reduced cell viability, and mobility by mediating PDPK1 in OS cells

Bannaxanthone E Induced Cell-Cycle Arrest and Apoptosis in Human Lung Cancer Cell Line

The anti-cancer activity of bannaxanthone E isolated from Garcinia mckeaniana leaves was assessed through a flow cytometric method on human lung SK-LU-1 cancer cells, including cell cycle changes and induction of cell apoptosis. Treatment with bannaxanthone E led to the suppression of cell cycle progression at the G2/M phase to 19.6% at 4 µM, and induced apoptosis via cell morphological changes, increased the fluorescence signal in caspase-3/7 activation, and accumulation of apoptotic cells in the SK-LU-1 line at 4 µM to 25.7%.

Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood

Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the proinflammatory cytokines, IL-1β and IL-18. Although inflammasomes are important for many diseases such as periodic fever syndromes, COVID-19, gout, sepsis, atherosclerosis and Alzheimer’s disease, only a little knowledge exists on the precise and cell type specific occurrence of inflammasome activation in patient samples ex vivo. In this report, we provide detailed information about the optimal conditions to reliably identify inflammasome activated monocytes by ASC speck formation using a modified flow cytometric method introduced by Sester et al. in 2015. Since no protocol for optimal sample processing exists, we tested human blood samples for various conditions including anticoagulant, time and temperature, the effect of one freeze–thaw cycle for PBMC storage, and the fast generation of a positive control. We believe that this flow cytometric protocol will help researchers to perform high quality translational research in multicenter studies, and therefore provide a basis for investigating the role of the inflammasome in the pathogenesis of various diseases.

Establishment of HLA class I and MICA/B null HEK-293T panel expressing single MICA alleles to detect anti-MICA antibodies

Pre- and post-transplantation anti-MICA antibody detection development are associated with an increased rejection risk and low graft survival. We previously generated HLA class I null HEK-293T using CRISPR/Cas9, while MICA and MICB genes were removed in this study. A panel of 11 cell lines expressing single MICA alleles was established. Anti-MICA antibody in the sera of kidney transplant patients was determined using flow cytometric method (FCM) and the Luminex method. In the 44 positive sera, the maximum FCM value was 2879 MFI compared to 28,135 MFI of Luminex method. Eleven sera (25%) were determined as positive by FCM and 32 sera (72%) were positive by the Luminex method. The sum of total MICA antigens, MICA*002, *004, *009, *019, and *027 correlation showed a statistically significant between the two methods (P = 0.0412, P = 0.0476, P = 0.0019, P = 0.0098, P = 0.0467, and P = 0.0049). These results demonstrated that HEK-293T-based engineered cell lines expressing single MICA alleles were suitable for measuring specific antibodies against MICA antigens in the sera of transplant patients. Studies of antibodies to MICA antigens may help to understand responses in vivo and increase clinical relevance at the cellular level such as complement-dependent cytotoxicity.

Establishment of HLA class I and MICA/B null HEK-293T panel expressing single MICA alleles to detect anti-MICA antibodies

Pre- and post-transplantation anti-MICA antibody detection development are associated with an increased rejection risk and low graft survival. We previously generated HLA class I null HEK-293T using CRISPR/Cas9, while MICA and MICB genes were removed in this study. A panel of 11 cell lines expressing single MICA alleles was established. Anti-MICA antibody in the sera of kidney transplant patients was determined using FCM (flow cytometric method) and the Luminex method. In the 44 positive sera, the maximum FCM value was 2,879 MFI compared to 28,135 MFI of Luminex method. Eleven sera (25%) were determined as positive by FCM and 32 sera (72%) were positive by the Luminex method. The sum of total MICA antigens, MICA*002, *004, *009, *019, and *027 correlation showed a statistically significant between the two methods (P = 0.0412, P = 0.0476, P = 0.0019, P = 0.0098, P = 0.0467, and P = 0.0049). These results demonstrated that HEK-293T-based engineered cell lines expressing single MICA alleles were suitable for measuring specific antibodies against MICA antigens in the sera of transplant patients. Studies of antibodies to MICA antigens may help to understand responses in vivo and increase clinical relevance at the cellular level such as complement-dependent cytotoxicity.

COMPARISON BETWEEN FLOW CYTOMETERIC METHOD AND OTHER METHODS FORRETICULOCYTE COUNT IN DIAGNOSIS OF PHENYLHYDRAZINE-INDUCED HEMOLYTIC ANEMIA

Reticulocyte count is the salient evidence of the effectiveness of bone marrow to produce red blood cells. Currently, the reticulocyte counting is a challenge for clinical laboratoriesmainly for the ordinary ones, which still use the manual method.This study was designed to evaluate the performance of flow cytometer for reticulocytes counting comparing to traditional and optimized manual methods which helpful in diagnosis of phenylhydazine-induced anemia.For that 45 male white Albino rats were divided into 5 groups, control group,phenylhydrazine group (PHZ) which injected by phz(20 mg/kg b.w, I/P),quercetin+phz group (quercetin, 50 mg/kgb.w per os), silymarin+phz group (silymarin, 100 mg/kgb.w per os) and quercetin group. Whole blood samples of these groups were collected at day 3, 5 and 10 after 1st injection of phz which used for reticulocyte counts by flow cytometeric method and other manual methods in addition to measurement ofCBC and osmotic fragility. Analysis of the results showed that phenylhydrazine injection induced hemolytic anemia with significant reticulocytosis and using of flow cytometer in reticulocyte count more precise, easy and fast than traditional and optimized manual methods. Furthermore, degree of hemolysis was significantly increases in phz group comparing to other groups. Therefore, we concluded that flow cytometric method for reticulocyte counts was simple, fast and highly reliable comparable to traditional and optimized manual methods. Also optimized manual showed that more perfect than traditional manual method and nearly to accuracy of flow cytometeric method.

Sex selection techniques and their implications.

Sex selection techniques provide economic benefits to dairy and beef herd management. Therefore, the development of such techniques has attracted the attention of reproductive biologists. There have been numerous studies concerning the development of sex selection techniques. As the sex of the offspring is determined by certain chromosomes, namely the X and Y chromosomes in mammals, most studies have focused on sperm sexing, attempting to separate the X and Y chromosome-bearing sperm based on differences in deoxyribonucleic acid (DNA) content, head size/volume, motility, and immunological specificity. However, most of these methods have failed to show reproducibility. Only the flow cytometric method has been confirmed to be accurate and reliable thus far. More than three decades have passed since this technique was first developed. The sexed semen produced with the method is currently commercialized and widely used for artificial insemination (AI) in cattle around the world. Recently, however, another technique based on differences in DNA contents using a fluidics device was developed by a commercial company. Studies focused on immunological approaches and the modification of sperm motility have also described the successful separation of the two types of sperm. Therefore, the present review evaluated the available sex selection techniques and their implications.