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Algologia ◽  
2021 ◽  
Vol 31 (4) ◽  
pp. 353-364
Author(s):  
N.A. Chernobai ◽  
◽  
K.D. Vozovik ◽  
N.G. Kadnikova ◽  
◽  
...  

The possibility of using various methods for determining the viability of cultures of microalgae Dunaliella salina and Chlorococcum dissectum before and after freezing-warming was investigated and analyzed. It has been established that the selection of an effective method should be carried out individually for each culture. For an integral assessment of the proliferative and metabolic activity of cells of both species of the studied microalgae, Alamar Blue-test and the ability to grow on liquid nutrient media can be used. The use of the Koch plate method, MTT-test and TTC staining is possible only for the microalga C. dissectum. Vital staining with trypan blue was found to be incorrect.


2021 ◽  
Vol 12 (4) ◽  
Author(s):  
L. V. Kladnytska ◽  
◽  
A. Y. Mazurkevych ◽  
S. V. Velychko ◽  
L. V. Garmanchuk ◽  
...  

The studies were conducted on 2–3-months-old males of C57BL/6 mice weighing 20–24 g. Obtaining and operating with adipose tissue-derived mesenchymal stem cell (MSC) culture was performed in a sterile laminar box under conditions of asepsis and antiseptics. The adipose tissue-derived MSC of the 2, 4, 7 and 12 passages were analyzed. Morphometric analysis was performed using a light microscopy. Morphometric parameters such as cell and nucleus area or nuclear-cytoplasmic ratio were calculated using the Axiovision light microscope (Carl Zeiss, Germany) and Image J 1.45 software. Trypan blue dye used for investigation of the viability of MSC. The morphological characteristics of adipose tissue-derived MSC during the process of cultivation changes: at the first passages of cultivation, the cells are spindle-shaped with two, at least three, long cytoplasmic processes, which are located bipolar. Near the nucleus, the Golgi complex is clearly visible – a sign of active cells. At later passages, cells have a small cytoplasmic processes and the bipolar arrangement of processes changes by stellar arrangement. Golgi complex is also clearly visualized. The indicator of the nuclear-cytoplasmic ratio in MSC from adipose tissue is significantly reduced at the 7th passage to 0.2189 ± 0.0122 (P < 0.01), and at the 12th passage to 0.1111 ± 0.0086 (P < 0.001) compared to the 2nd passage. The coefficient of proliferation of adipose tissue-derived MSC is significantly reduced at 12th passage. The viability of MSC from adipose tissue with an increasing of a number of passages significantly reduces and at the 12th passage of cultivation reaches 84.67 ± 1.36 (P < 0.05). The content of apoptotic cells that exhibited sensitivity to serum-free cultivation significantly increased at the 7th and 12th passages and was 21.33 ± 1.36 (P < 0.05) and 23.67 ± 0.97% (P < 0.05), respectively.


2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Burak Durmaz ◽  
Bakiye Goker Bagca ◽  
Ozgur Cogulu ◽  
Sunde Yilmaz Susluer ◽  
Araz Alpay ◽  
...  

Prednisolone has been used frequently in the treatment of acute lymphoblastic leukemia. However, to overcome the challenges of the treatment, the development of additional therapies is of great importance. Small, non-protein-coding RNAs, namely, microRNAs (miRNAs), are critical epigenetic regulators with physiological and pathological importance. This study is aimed at determining the effects of miR-146a, miR-155, and miR-181a inhibition with their corresponding anti-miRs on both leukemic and healthy cells, individually and with prednisolone. Leukemic (SUP-B15) and healthy B-lymphocyte (NCI-BL 2171) cell lines were used in this study. A total of 12 experimental groups included individual and combinational silenced ALL-associated miRNAs (hsa-miR-155, hsa-miR-146a, and hsa-miR-181a) and their combination with prednisolone. Cytotoxicity, proliferation, cell cycle, and apoptosis analyses were performed by using WST-1, trypan blue, APC-BrdU, Annexin V, and JC-1 methods in each study group, respectively. To control the effectiveness of anti-miR transfection and prednisolone application, miRNA expression analysis was performed from all groups. Anti-miR application was effective on the viability, proliferation, cell cycle, and apoptosis of leukemia cells, and this effect was increased with prednisolone administration. In addition, this activity was found to be very low on healthy cells. In conclusion, anti-miR applications may have the potential for clinical use of adjuvant to or as an alternative to conventional therapies for childhood acute lymphoblastic leukemia.


Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1468
Author(s):  
Shakirat A. Adetunji ◽  
Dmitriy Smolensky ◽  
Dana N. Mitzel ◽  
Jeana L. Owens ◽  
Carol G. Chitko-McKown ◽  
...  

Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne pathogen that regularly causes severe neurological disease in humans in Southeast Asia and the Western Pacific region. Pigs are one of the main amplifying hosts of JEV and play a central role in the virus transmission cycle. The objective of this study was to identify in vitro cell systems to investigate early effects of JEV infection including viral replication and host cell death. Here, we demonstrate the susceptibility of several porcine cell lines to the attenuated genotype III JEV strain SA14-14-2. Monolayers of porcine nasal turbinate (PT-K75), kidney (SK-RST), testis (ST), and monocyte-derived macrophage (CΔ2+) cells were infected with SA14-14-2 for up to five days at a multiplicity of infection (MOI) of 0.1. The hamster kidney cell line BHK-21, previously shown to be susceptible to SA14-14-2, was used as a positive control. Culture supernatants and cells were collected between 0 and 120 h post infection (hpi), and monolayers were observed for cytopathic effect (CPE) using brightfield microscopy. The number of infectious virus particles was quantified by plaque assay and cell viability was determined using trypan blue staining. An indirect immunofluorescence assay was used to detect the presence of JEV NS1 antigens in cells infected at 1 MOI. All four porcine cell lines demonstrated susceptibility to SA14-14-2 and produced infectious virus by 12 hpi. Virus titers peaked at 48 hpi in CΔ2+, BHK-21, and SK-RST cells, at 72 hpi in PT-K75, and at 120 hpi in ST cells. CPE was visible in infected CΔ2+ and BHK-21 cells, but not the other three cell lines. The proportion of viable cells, as measured by trypan blue exclusion, declined after 24 hpi in BHK-21 and 48 hpi in CΔ2+ cells, but did not substantially decline in SK-RST, PT-K75 or ST cells. At 48 hpi, JEV NS1 was detected in all infected cell lines by fluorescence microscopy. These findings demonstrate several porcine cell lines which have the potential to serve as useful research tools for investigating JEV infection dynamics and host cell mechanisms in a natural amplifying host species, such as pigs, in vitro.


2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A712-A712
Author(s):  
Randy Tsai ◽  
Hannah Fields ◽  
Xinlian Zhang ◽  
Valentina Ferrari ◽  
Soo Park ◽  
...  

BackgroundMyelodysplastic syndromes (MDS) are the most common acquired cause of bone marrow failure. Though DNA hypomethylating agents (HMAs) such as 5-Azacitidine (5-Aza) may increase survival of patients with higher-risk MDS, their mechanistic effects on hematopoiesis and immune cell function remain unclear. Using whole exome sequencing analysis, we previously identified MDS-related mutations within monocyte-derived dendritic cells (moDCs) from patients with higher-risk MDS. Here we examine the effect of 5-Aza on the phenotype of moDCs from the same cohort of patients with higher-risk MDS.MethodsPurified CD14+ cells were magnetically isolated from peripheral blood mononuclear cells from 6 patients with IPSS-R Intermediate/High/Very High-risk MDS (herein collectively referred to as higher-risk MDS). Cells were cultured in complete medium with IL-4 (800 U/mL) and GM-CSF (1200 U/mL) for 5 days. Freshly prepared 5-Aza or dimethylsulfoxide (DMSO) vehicle was added to cultures every 24 hours for a total of three 1 μM doses starting on Day 1. Immature moDCs were then stimulated with poly(I:C) (20 ng/mL), IL-1β (25 ng/mL), IFN-α (3000 U/mL), IFN-γ (1000 U/mL), and TNF-α (50 ng/mL) for 48 hours to generate moDCs. Flow cytometry analyses were performed with Guava easyCyte 8HT before and after addition of maturation cocktail.ResultsBased on trypan blue staining, in vitro addition of 5-Aza to CD14+ cells from 6 patients with higher-risk MDS did not result in a significant reduction in the percentage of cell survival on Day 5 and Day 7 in culture (figure 1a, p=0.8765 and p=0.7109, respectively). Treatment with 5-Aza significantly reduced the percentage of CD14-CD209+ moDCs on Day 7 following the addition of maturation cocktail (figure 1b, p<0.0001). Flow cytometry assessment showed comparable expression of common maturation and co-stimulatory markers such as CD80, CD83, CD86, HLA-DR, CD209, CD141, CD40, and CCR7 between 5-Aza and DMSO-treated immature moDCs on Day 5 (figure 1c). Similarly, 5-Aza treatment had no significant effect on marker expression on mature moDCs generated with maturation cocktail on Day 7.ConclusionsThere was no significant difference in maturation and co-stimulatory marker expression of immature and mature moDCs from patients with higher-risk MDS following in vitro treatment with 5-Aza. Though recent studies have identified important immunoregulatory effects of 5-Aza, functional changes that may occur within the dendritic cell population are not fully understood. Further studies are planned, including cytokine analyses and transcriptome sequencing of mature moDCs, and may help elucidate the immunological mechanisms underlying the therapeutic effects of 5-Aza in patients with higher-risk MDS.Ethics ApprovalThe study is being conducted as per the Declaration of Helsinki and was approved by the University of California San Diego Institutional Review Board (#161345) and registered with ClinicalTrials.gov (NCT02667093). All patients were provided written informed consent.Abstract 684 Figure 15-Aza and DMSO vehicle-treated moDCs from patients with higher-risk MDS were evaluated for phenotypic markers before and after stimulation with maturation cocktail. Purified CD14+ cells were magnetically isolated from PBMC from 6 higher-risk MDS patients and cultured with IL-4 and GM-CSF for 5 days followed by addition of poly(I:C), IL-1β, IFN-α, IFN-γ, and TNF-α for 48 hours at 37°C in a 5% CO2 incubator. Freshly prepared 5-Aza or DMSO vehicle was added to cultures every 24 hours for a total of three 1 μM doses starting on Day 1. (A) Cultured cells were stained with trypan blue to determine the percentage of cell survival on Day 5 and Day 7 in culture. (B) Treatment with 5-Aza significantly reduced the percentage of CD14-CD209+ moDCs on Day 7 following addition of maturation cocktail (p<0.0001). (C) The percentage of CD14-CD83+ cells is comparable between 5-Aza and vehicle-treated immature moDCs on Day 5 and mature moDCs on Day 7 (p=0.2434 and p=0.5846, respectively). (D) Cultured cells were stained with fluorochrome-conjugated antibodies to determine the expression of common maturation and co-stimulatory markers using flow cytometry. Cells were gated on CD14-CD11c+ to distinguish moDCs, and scatterplots represent the geometric mean fluorescence intensity (gMFI) of marker expression pre- and post-maturation. Individual dots represent one of three experimental replicates performed for the 6 higher-risk MDS patient samples. Each dot is labeled by MDS patient sample. Statistical analysis was performed by Welch's t-test using GraphPad Prism.


2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Qunfeng Wu ◽  
Zheng Feng ◽  
Wei Hu

Abstract Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.


Animals ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2934
Author(s):  
Yang He ◽  
Erlong Wang ◽  
Kaiyu Wang ◽  
Jun Wang ◽  
Wei Fan ◽  
...  

The spleen is a separate organ of the teleost, playing an essential role in immune reactions. The morphology of the spleen is different from the fish species. Little knowledge about the spleen structure and the blood splenic barrier (BSB) in Nile tilapia has been reported. To address this issue, we studied the histology of the spleen and the BSB in healthy Nile tilapia. The morphology of the spleen was observed, then H&E staining, modified Jame’s staining, and ultrastructural techniques were performed to portion the spleen into three subregions and analyze the location of components and fibers. Thereafter, vital staining of Nile tilapia with Trypan blue was conducted to elucidate the composition and function of BSB. Histologically, the spleen could be divided into three subregions (inner, middle, and outer). The venules, clumps of lymphocytes, and vessels were separately characterized features of the outer, middle, and inner layers. Post injection, Trypan blue was intercepted in the endotheliocytes of ellipsoids in the middle layer (i.p.) or was deposited to the reticular fibers surrounding the ellipsoids (i.v.). Additionally, the amount of Trypan blue was shown to be positively correlated to that of the Acid phosphatase expressed. In conclusion, the spleen could be portioned into three subregions, and the BSB lay in the middle layer, composed of the cuboidal-shaped endotheliocytes and the surrounding reticular fibers of the ellipsoid capillaries. The present study enriched the research of immune tissues and system in tilapia and provided reference for the study of spleen in other fish species.


2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Giuseppina Bozzuto ◽  
Giuseppe D’Avenio ◽  
Maria Condello ◽  
Simona Sennato ◽  
Ezio Battaglione ◽  
...  

Abstract Background There is a huge body of literature data on ZnOnanoparticles (ZnO NPs) toxicity. However, the reported results are seen to be increasingly discrepant, and deep comprehension of the ZnO NPs behaviour in relation to the different experimental conditions is still lacking. A recent literature overview emphasizes the screening of the ZnO NPs toxicity with more than one assay, checking the experimental reproducibility also versus time, which is a key factor for the robustness of the results. In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of multiple independent assays. Results ECIS-measurements were compared with traditional cytotoxicity tests such as MTT, Neutral red, Trypan blue, and cloning efficiency assays. ECIS could follow the cell behavior continuously and noninvasively for days, so that certain long-term characteristics of cell proliferation under treatment with ZnO NPs were accessible. This was particularly important in the case of pro-mitogenic activity exerted by low-dose ZnO NPs, an effect not revealed by endpoint independent assays. This result opens new worrisome questions about the potential mitogenic activity exerted by ZnO NPs, or more generally by NPs, on transformed cells. Of importance, impedance curve trends (morphology) allowed to discriminate between different cell death mechanisms (apoptosis vs autophagy) in the absence of specific reagents, as confirmed by cell structural and functional studies by high-resolution microscopy. This could be advantageous in terms of costs and time spent. ZnO NPs-exposed A549 cells showed an unusual pattern of actin and tubulin distribution which might trigger mitotic aberrations leading to genomic instability. Conclusions ZnO NPs toxicity can be determined not only by the intrinsic NPs characteristics, but also by the external conditions like the experimental setting, and this could account for discrepant data from different assays. ECIS has the potential to recapitulate the needs required in the evaluation of nanomaterials by contributing to the reliability of cytotoxicity tests. Moreover, it can overcome some false results and discrepancies in the results obtained by endpoint measurements. Finally, we strongly recommend the comparison of cytotoxicity tests (ECIS, MTT, Trypan Blue, Cloning efficiency) with the ultrastructural cell pathology studies. Graphic Abstract


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