Cells In 96-well Plates Research Articles

Screening persistent organic pollutants for effects on testosterone and estrogen synthesis at human-relevant concentrations using H295R cells in 96-well plates.

Many persistent organic pollutants (POPs) are suspected endocrine disruptors and it is important to investigate their effects at low concentrations relevant to human exposure. Here, the OECD test guideline #456 steroidogenesis assay was downscaled to a 96-well microplate format to screen 24 POPs for their effects on viability, and testosterone and estradiol synthesis using the human adrenocortical cell line H295R. The compounds (six polyfluoroalkyl substances, five organochlorine pesticides, ten polychlorinated biphenyls and three polybrominated diphenyl ethers) were tested at human-relevant levels (1 nM to 10 µM). Increased estradiol synthesis, above the OECD guideline threshold of 1.5-fold solvent control, was shown after exposure to 10 µM PCB-156 (153%) and PCB-180 (196%). Interestingly, the base hormone synthesis varied depending on the cell batch. An alternative data analysis using a linear mixed-effects model that include multiple independent experiments and considers batch-dependent variation was therefore applied. This approach revealed small but statistically significant effects on estradiol or testosterone synthesis for 17 compounds. Increased testosterone levels were demonstrated even at 1 nM for PCB-74 (18%), PCB-99 (29%), PCB-118 (16%), PCB-138 (19%), PCB-180 (22%), and PBDE-153 (21%). The MTT assay revealed significant effects on cell viability after exposure to 1 nM of perfluoroundecanoic acid (12%), 3 nM PBDE-153 (9%), and 10 µM of PCB-156 (6%). This shows that some POPs can interfere with endocrine signaling at concentrations found in human blood, highlighting the need for further investigation into the toxicological mechanisms of POPs and their mixtures at low concentrations relevant to human exposure.

Investigating the effect of astaxanthin on the survival rate of MCF-7 breast cancer cells by MTT method (cytotoxicity test)

Cancer is known as a very dangerous and incurable disease, with symptoms such as pain and disability. In recent years, changes in the way people live have increased the prevalence of breast cancer in different societies. This terrible disease has increased research in this field. Astaxanthin is one of the most famous natural carotenoids, which, due to its strong antioxidant activity, can prevent cardiovascular diseases, strengthen the body's immune system, and prevent cell ageing. In this study, the effect of astaxanthin on MCF-7 cancer cells was investigated. After culturing the cells in 96-well plates, they were treated with different concentrations of astaxanthin and then evaluated using the MTT method. The results showed that astaxanthin can inhibit cell growth and induce apoptosis, and this effect is enhanced by increasing the concentration of astaxanthin, so that at a concentration of 100 μM, the rate of apoptosis increased to 35%. Therefore, it can be concluded that astaxanthin may be used as a control treatment for cancer.

Abstract 3976: Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach

Abstract Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods. Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) ɑ/β or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these ɑ/β and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Lester Kobzik, Khadija Ghias, Paul Diehl. Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3976.

Tangeretin's Anti-apoptotic Signaling Mechanisms in Oral Cancer Cells: In Vitro Anti-cancer Activity.

Introduction Citrus fruit peels contain Tangeretin, a natural chemical flavonoid that reinforces plant cell walls and serves as a defense mechanism. Apoptosis, growth inhibition, anti-oxidant, anti-diabetic, and anti-cancer activities are only a few of its many qualities. Tangeretin's principal function is to shield healthy cells or tissues from the harmful effects of chemotherapy. The purpose of this study was to investigate the apoptotic activity of Tangeretin's impact on KB (oral cancer cells) cell lines. Materials and method This study employed Tangeritin, in investigating its effects on oral cancer cells. Oral cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM), with 10% fetal bovine serum at 37°C in a 5% CO2 environment. Cell viability was assessed by seeding oral cancer cells in 96-well plates, exposing them to varying Tangeritin concentrations (50 µM, 100 µM, and 200 µM) with growth inhibition of KB cell viability in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayand morphological changes in cells were observed under an inverted light microscope at 10x magnification. The results were reported as mean±standard error mean (SEM) using one-way analysis of variancethrough IBM SPSS Statistics for Windows, Version 23 (Released 2015; IBM Corp., Armonk, New York, United States). Result MTT assay showed a significant reduction in KB cell viability when treated with Tangeretin. With asignificant decrease in mRNA levels of the anti-apoptotic genes Bcl-2 and Bcl-xL. At 50 µM, 100 µM, and 200 µM, the levels of Bcl-2 were 0.85 ± 0.09, 0.62 ± 0.05, and 0.67 ± 0.05, respectively. Similarly, the mRNA expression of Bcl-xL was 0.82 ± 0.07 for 50 µM, 0.7 ± 0.06 for 100 µM, and 0.77 ± 0.06for 200 µM. The mRNA expression levels of Bax were 1.1 ± 0.09 for 50 µM, 1.4 ± 0.12for 100 µM, and 1.3 ± 0.11 for 200 µM, respectively. Conclusion Tangeretin showed a promising apoptotic activity in KB cells suggesting its utility as an anti-cancer compound. It prevented the growth and proliferation of cancer cells by acting on pro-apoptotic and anti-apoptotic genes. However, this conclusion is mostly based on the in vitro study. Therefore in vivo animal studies were needed to confirm the findings.

Cytotoxicity Test of Active Compounds Natural Ingredients of Snail Mucus (Achatina fulica) Against BHK-21 Fibroblast Cells

Snails are unpleasant yet beneficial. Rural people have used one to treat illnesses like toothache for years. We will test snail's mucus Achatina fulica's cytotoxic activity against Baby Hamster Kidney (BHK-21) fibroblast cells at 12.5%, 25%, 50%, 100% and its resistance to Phorpyromonas gingivalis, Fusobacterium nucleatum, E. Faecalis, and S. aureus using Microtetrazolium (MTT) assay. The test and comparison solution was incubated with 5x103/100 l cells in 96-well plates. 5 mg/mL MTT completed the solution's incubation. ELISA readers measured purple color intensity. The formula transformed absorbance data at 595 nm into percent alive cells. ELISA readers read data. ANOVA, parametric Kolmogorov-Smirnov data normality test were performed. The cytotoxicity statistical test shows the following results: 12.5 % (0.76875 ±0.01117), 25% (0.49350 ±0.004796), 50% (0.30250 ±0.006658) and 100% (0.171 ±0.10488). The lowest cytotoxicity of Achatina fulica snail mucus is 12.5% with an average of 0.768. Achatina fulica snail mucus resists Phorpyromonas gingivalis, Fusobacterium nucleatum, E. Faecalis, and S. aureus at 12.5%.

High-throughput Quantification of Antibody-Dependent Phagocytosis using Live-Cell Analysis

Abstract The uptake and clearance of tumor cells can be promoted with monoclonal antibodies (mAbs) via antibody-dependent phagocytosis (ADCP) or through the blockade of “don’t-eat-me” signals, such as CD47. These mechanisms hold immunotherapeutic promise and are studied extensively in drug development. High-throughput assays were conducted using pHrodo®, a pH-sensitive fluorophore, that enables live-cell imaging and kinetic quantification of phagocytosis. pHrodo® labeled target cells were incubated with mAbs and added to effector cells in 96-well plates. Images were acquired using the Incucyte® Live-Cell Analysis System and fluorescence automatically quantified with integrated software. In line with known pro-phagocytic effects, anti-CD47 promoted phagocytosis of CCRF-CEM tumor cells by blocking CD47 “don’t-eat-me” signals. Clinical anti-CD20 mAbs Truxima® and Rituximab both promoted ADCP of Ramos target cells by primary macrophages. In addition, the ADCP response of different Rituximab isotypes were compared. Anti-CD20-IgG1 mAb and Fc mutated anti-CD20-IgG1fut (non-fucosylated) exhibited concentration-dependent responses (EC50 values of 25.1 ng/mL and 42.8 ng/mL, respectively), however anti-CD20-IgG1NQ (non-glycosylated) showed no response. ADCP was also examined in adherent target cells with varied HER2 profiles. Anti-HER2 (Trastuzumab) induced an ADCP response in HER2-positive AU565 cells but not in HER2-low MDA-MB-231 cells, consistent with established correlations between HER2 expression and ADCP response. These data exemplify that live-cell analysis is a powerful approach that enables functional quantification of ADCP and is amenable to antibody screening for therapeutic candidates.

Addition of arginine hydrochloride and proline to the culture medium enhances recombinant protein expression in Brevibacillus choshinensis: The case of RBD of SARS-CoV-2 spike protein and its antibody

Brevibacillus choshinensis is a gram-positive bacterium that is known to efficiently secrete recombinant proteins. However, the expression of these proteins is often difficult depending upon the expressed protein. In this study, we demonstrated that the addition of arginine hydrochloride and proline to the culture medium dramatically increased protein expression. By culturing bacterial cells in 96-well plates, we were able to rapidly examine the expression conditions and easily scale up to 96 mL of culture for production. Although functional expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein without any solubility-enhancing tag in bacterial strains (including Escherichia coli) has not been reported to date, we succeeded in efficiently producing RBD which showed a similar CD spectrum to that of RBD produced by eukaryotic cell expression systems. Furthermore, RBD from the omicron variant (B.1.1.529) was also produced. Physicochemical analyses indicated that omicron RBD exhibited markedly increased instability compared to the wild-type. We also revealed that the Fab format of the anti-SARS-CoV-2 antibody C121 can be produced in large quantities using the same expression system. The obtained C121 Fab bound to wild-type RBD but not to omicron RBD. These results strongly suggest that the Brevibacillus expression system is useful for facilitating the efficient expression of proteins that are difficult to fold and will thus contribute to the rapid physicochemical evaluation of functional proteins.

Radiation Induced and FACS-Sorted Senescent tdTOMp16+ Cells Upregulate Profibrotic Gene Expression in Mesenchymal Stem Cells (Stromal Cells)

The role of cellular senescence in radiation induced pulmonary fibrosis (RIPF) is unknown. Furthermore, clearance of senescent cells (SCs) could be an effective therapeutic strategy; however, the identification and targeted removal of only senescent cells by senolytic drugs is difficult. We isolated a pure population of radiation induced senescent and non-senescent cells using tdTOMp16+ mice and examined induction of fibrosis in target cells.A stromal cell line was established from explanted tdTOMp16+ mouse bone marrow. The tdTOMp16+ stromal cell line was irradiated to each of several doses by Cesium Gamma Cell dose rate 300 cGy/min including 1.0 Gy, 2.5 Gy, 5 Gy, and 7.5 Gy under growth conditions of either 50% or 100% confluence. The number and percent of senescent cells, by p16 promoter linked to the red fluorochrome biomarker was quantitated. Irradiated red, irradiated non-red, and non-irradiated cells were sorted and cultured as single cells in 96-well plates, then monitored for cell division over four weeks. The expression of senescence markers, p16 and p21, was evaluated by qPCR, and SA-β-gal by fluorogenic substrate. To examine the effect of senescent cells on a target fibrosis-prone C57BL/6 line, sorted irradiated red, irradiated non-red, and non-irradiated cells were placed on the top level of transwells, and after 2 weeks induction of biomarkers of fibrosis including Ctgf, TGF-β, collagen 1a, and collagen 3 was measured in target cells in the lower level by qPCR.After 5 Gy irradiation at 50% cell density, ∼9% of tdTOMp16+ cells became senescent (stably red in color) after 10 days, thus, defining the optimal conditions. Then, in bulk cultures, red cells were sorted by fluorescence automated cell sorter (FACS) to a pure population of red tdTOMp16+ cells. When cultured as single cells, none of 960 irradiated red cells divided after 2 weeks, 137 of these cells remained intact, and assume large and flat morphology. In contrast, 50 of 800 non-red irradiated cells divided, and 104 of 560 non-irradiated control cells divided. There was significant detection of senescent cell markers including: SA-β-gal, p16, and p21 in irradiated sorted red cells compared to irradiated non-red cells (P = 0.0096, P < 0.0001 and P = 0.0001 respectively based on scoring of 1000 cells in triplicate experiments for SA-β-gal and analyses from three independent experiments). Induction of biomarkers of fibrosis Ctgf, TGF-β, collagen 1a, and collagen 3 in C57BL/6 stromal target cells was significant by irradiated red, but not irradiated non-red or non-irradiated cells (P = 0.037, P = 0.14, P = 0.015, and P = 0.0259).The data indicate that sorted irradiated red senescent tdTOMp16 stromal cells can be isolated as a pure non-dividing population and induce fibrotic markers in target cells in non-contact transwell cultures. This unique system can now be used to define the mechanism of senescent cell-induced radiation fibrosis, and the effects of senolytic drugs.

Demonstration of a Single-Parameter, Rapid Flow Cytometry Assay in Assessing Natural Killer Cell Cytotoxic Function

Assessing cytotoxic function of immune effector cells, such as natural killer (NK) cells, is necessary to determine their potency. Various cytotoxicity assays measuring target cell death have been reported, however these assays typically feature a number of limitations that curb their broad applicability. Quantitative assays of NK cell killing traditionally measure the release of chromium or a fluorescent dye from labelled target cells. These assays typically show low reproducibility and often underestimate cell death for many cell types. Recent strategies aimed at ensuring manufacture of high NK cell doses commonly include NK cell co-culture with antigen presenting cells as feeder cells for their expansion. Such expansion platforms result in a final NK cell product with a high content of dead or dying feeder cells. This end product also complicates the accurate measurement of target cell death in NK cell cytotoxicity assays. Here we report a rapid flow cytometry assay for quantifying target cell killing after single-color labeling of the target cells prior to co-incubation with NK cells. In this assay, target cells are first labeled with calcein-AM (CAM), a green fluorescent dye. CAM-labeled target cells are then co-incubated with feeder cell expanded-NK cells in 96-well plates using various NK cell-to-target ratios. After 4 hours of NK cell-target cell co-incubation, a fixed volume of the cell suspension was analyzed using a flow cytometer capable of reading 96-well plates. The live, CAM-labeled target cells were gated and quantitated for all the treatment conditions to determine the number of CAM-labeled target cells present per fixed volume.Using this assay, we observed highly consistent cytotoxicity results against both suspension and adherent cell targets. The accuracy of this assay was also demonstrated by NK cell dose-dependent responses. Importantly, the presence of dead feeder cells in the NK cell suspension does not interfere with the analysis of this assay. In addition, the gating strategy is simple, consistent, and the analysis between treatment groups does not require cumbersome calculations or compensation that could introduce variability. Assessment of target cell death was also shown to be more reliable and consistent when compared with the measurement of CAM release using a microplate reader. The latter assay has previously been shown to be highly variable with inconsistent results depending on target cell and CAM dilution. Our flow cytometry assay consistently identifies live cells, avoiding errors due to variability in dye intake and dye release detection. DisclosuresNo relevant conflicts of interest to declare.

Abstract PO-025: Radiation-induced and FACS-sorted senescent tdTOMp16+ cells upregulate profibrotic genes in C57BL/6 stromal target cells

Abstract Acute Radiation Syndrome and the multiorgan failure from delayed effects of acute radiation exposure present challenging consequences of radiation terrorism or a radiological accident. Recent research suggests that radiation-induced cellular senescence plays an important role in radiation-induced pulmonary fibrosis (RIPF), and that clearance of senescent cells (SCs) could be an effective therapeutic strategy. However, the identification and targeted removal of only senescent cells using drugs have been difficult. Here we have established a bone-marrow stromal cell line from a tdTOMp16+ mouse. We show that 5 Gy irradiation induces ~9% cellular senescence in tdTOMp16+ stromal line after 10 days and can be isolated as a pure population of red tdTOMp16+ cells by FACS sorting. To confirm that these irradiated and then sorted red cells are senescent, we cultured them as single cells in 96-well plates and followed for four weeks. None of the 960 irradiated red cells we plated divided after 2 weeks, 137 of these cells remained intact and assumed large and flat cellular morphology. In contrast, 50 of 800 cells that were irradiated but didn’t turn red, divided, as did 104 of 560 nonirradiated control cells (0 Gy) divided. There was significant upregulation of senescent cell markers including SA-ß-gal, p16, and p21 in the irradiated and sorted red cells when compared to irradiated nonred cells. Sorted irradiated red, non-irradiated, or irradiated non-red cells were placed on the top well of transwells. There was a significant induction of fibrotic genes Ctgf, Tgf- ß, collagen 1a, and collagen 3 in C57/B6 stromal target cells in the bottom layer by irradiated red cells, but not the other cell populations irradiated non-red. Thus, radiation-induced biomarkers of fibrosis are induced by senescent cells. Citation Format: Amitava Mukherjee, Michael Epperly, Donna Shields, Wen Hou, Renee Fisher, Diala Hamade, Joel S. Greenberger. Radiation-induced and FACS-sorted senescent tdTOMp16+ cells upregulate profibrotic genes in C57BL/6 stromal target cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-025.

The edge effect: A global problem. The trouble with culturing cells in 96-well plates

BackgroundThe use of 96-well plates is ubiquitous in preclinical studies. Corner and edge wells have been observed to be more prone to evaporation compared to interior wells. MethodsMammalian cells were cultured in 96-well plates over a period of 72 h. VWR and Greiner plates were tested. MTS reagent was added, and metabolic activity was determined after 2 h. ResultsWhen using VWR plates, cells showed a highly heterogeneous pattern of cell growth. The outer wells showed 35% lower metabolic activity than the central wells. Cells grown in rows two and three also grew sub-optimally (25% and 10% reduction compared to central wells). Greiner plates showed better homogeneity. Cells grown in the outer wells showed 16% lower metabolic activity while cells in rows two and three showed reductions of 7 and 1%, respectively. This edge effect was partially mitigated by storing the plates in loosely sealed wrapping during incubation. Placing a buffer between the wells of the plate further improved homogeneity for the Greiner plates. ConclusionDifferent brands of 96-well plates show different levels of the edge effect. Some clearly are inappropriate for such studies. General significanceEach laboratory needs to determine their own optimum conditions for culturing cells empirically before continuing to use multiwell plates. Otherwise, large artifacts may arise, affecting the quality of data, with the potential of introducing type I or type II errors.

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below −20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.

The neuroprotective mechanism of cinnamaldehyde against amyloid-β in neuronal SHSY5Y cell line: The role of N-methyl-D-aspartate, ryanodine, and adenosine receptors and glycogen synthase kinase-3β.

Cinnamaldehyde may be responsible for some health benefits of cinnamon such as its neuroprotective effects. We aimed to investigate the cinnamaldehyde neuroprotective effects against amyloid beta (Aβ) in neuronal SHSY5Y cells and evaluate the contribution of N-methyl-D-aspartate (NMDA), ryanodine, and adenosine receptors and glycogen synthase kinase (GSK)-3β, to its neuroprotective effects. After seeding the cells in 96-well plates, adenosine (20, 40, 80, and 120 µM), NMDA (20, 40, 80, and 120 µM), and dantrolene (as a ryanodine receptor antagonist; 2, 4, 6, 8, and 16 µM) were added to the medium containing Aβ25-35 and/or cinnamaldehyde. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide method was used to assess neurotoxicity and western blot to measure the GSK-3β protein level. Cinnamaldehyde (15, 20, 23, and 25 μM) significantly reversed Aβ-induced toxicity in SHSY5Y neuronal cells. Adenosine (20, 40, 80 and 120 μM) inhibited the neuroprotective effects of cinnamaldehyde (15 μM). NMDA (20, 40, 80, and 120 μM) reduced cinnamaldehyde (15 and 23 μM) neuroprotective effects against Aβ neurotoxicity. Dantrolene (2, 4, 8, and 16 μM) significantly reduced cinnamaldehyde (15 μM) neuroprotective effects. Cinnamaldehyde (15 and 23 μM) suppressed the Aβ-induced increment of GSK-3β protein level. NMDA and adenosine receptors suppression together with ryanodine receptors stimulation may be relevant to cinnamaldehyde neuroprotective effects against Aβ neurotoxicity. Moreover, the inhibition of GSK-3β may contribute to the cinnamaldehyde neuroprotection.

Novel Silver-NHC complexes: Synthesis and anticancer properties

The aim of this study was to present the synthesis, characterization and anticancer activities of two novel benzimidazole-based NHC salts (1a-b) and their silver(I) complexes (2a-b) with methylbenzyl and isopropylbenzyl chains. All compounds were prepared using Schlenk techniques in an inert atmosphere. The carbene complexes were prepared with the interaction of carbene precursors and Ag2O. All new compounds were characterized by elemental analysis, LC-MS, FT-IR, 1H NMR, and 13C NMR spectroscopic techniques. The anticancer activities of the salts and complexes were determined by cell proliferation analysis using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human prostate cancer cells (DU-145) and human breast cancer cells (MCF-7, MDA-MB-231). L-929, mouse adipose tissue fibroblasts were used as model normal cells. The cells were plated at a cell density of 1 × 105 cells in 96-well plates and treated with different concentrations (1–20 μM) of salts and complexes during 24, 48 and 72 h. The MTT assay results indicated that the salts (1a-b) have lower anticancer activity on cancer cells than their silver(I) complexes (2a-b). It was also observed that benzimidazole salts (1a-b) and their Ag-NHC complexes (2a-b) displayed lower anticancer activities on L-929 normal cells than on cancer cells. These results highlight that the anticancer activities of N-heterocyclic carbene-silver complexes vary according to the structure of the silver complex and cell line type.

Monitoring Non-vesicular Transport of Phosphatidylserine and Phosphatidylinositol 4-Phosphate in Intact Cells by BRET Analysis.

Non-vesicular lipid transport via lipid transfer proteins (LTPs) at membrane contact sites (MCSs) is critical for the maintenance of the lipid composition of biological membranes. The ability to measure lipid transfer activity of diverse LTPs in live cells without interrupting the fine structural organization is essential to better understand the contribution of non-vesicular lipid transport to membrane organization. Here, we describe a semiquantitative method to analyze phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine (PS) changes at the plasma membrane (PM) as they relate to LTP functions. This live cell method is based on bioluminescence resonance energy transfer (BRET) measurements between a luciferase-tagged lipid-recognizing module and a PM-targeted fluorescent acceptor. Oxysterol-binding protein-related protein (ORP) 5 is a PI4P/PS lipid transfer protein which is stably tethered to the ER and also dynamically interacts with PM PI4P/PI(4,5)P2 via its pleckstrin homology (PH) domain. We show that this method is able to detect PI4P and PS changes in the PM upon acute recruitment of an ORP5 construct to the PM. This method is convenient and robust, utilizing population of cells in 96-well plates analyzed in a plate reader. We will also highlight potential further applications extending the method for other LTPs and other lipid cargoes.

20 Generation of myostatin knockout horse embryos using clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 and somatic cell nuclear transfer

Historically, livestock improvement by selective breeding was the principal selection force in animal production and welfare, but the desired phenotype may involve more than 1 generation. Nowadays, new technologies such as CRISPR/Cas9 could overpass these limits and improve animal quality by insertion or modification of the desired genotype. In this work, we aim to knock out the myostatin (MSTN) gene, a negative regulator of muscle mass development, in equine cells and to generate equine cloned embryos with this modified genotype. To achieve this, 1×105 equine fibroblasts were nucleofected with 5, 2, or 1μg of the plasmid hspCas9-2A-Puro V2.0 that codified for the Cas 9 nuclease and a single guide RNA (gRNA). Two different gRNA (gRNA 1 and gRNA 2) complementary to the first exon of the MSTN gene were designed and evaluated. Cells were also nucleofected with the enhanced green fluorescent protein-N1 plasmid in order to determine the transfection efficiency, obtaining more than 90% of enhanced green fluorescent protein+ cells in the 3 conditions. Forty-eight hours after nucleofection, cells were treated with 2.5μg mL−1 of puromycin for 48h to isolate cells that incorporated the plasmid. After that, clonal culture was achieved by plating individual cells in 96-well plates. The clones were then expanded individually and genomic DNA was isolated from each one, genotyped for the MSTN exon 1 locus by PCR amplification, and Sanger sequenced. Both gRNA had mutational activity, with 96% efficiency (24/25 clones) for gRNA 1 and 55.5% mutation activity (10/18 clones) for gRNA2. We obtained different genotypes depending on the gRNA and the dose that was used-gRNA 1: 1μg=57% wt/wt, 14% wt/mutX, and 29% mutX/mutX; 2μg=33.3% wt/wt, 33.3% mutX/mutY, and 33.3% mutX/mutX; 5μg=40% wt/wt and 60% mutX/mutY; gRNA 2: 1μg=17% wt/wt, 17% wt/mutX, 50% mutX/mutY, and 17% mutX/mutX; 2μg=67% mutX/mutY and 34% mutX/mutX; 5μg=54% mutX/mutY and 46% mutX/mutX. Two of the gRNA2 mutated cell lines were used for embryo generation by NT, 1 wt/mutX line (gRNA2-1ug-C2, heterozygote clone) and 1 mutX/mutX line (gRNA2-5ug-C13, homozygote clone). Before that, to assess specificity, the first 2 highly ranked off-target sites of gRNA2 were checked by Sanger sequencing in the selected clones, not observing modifications in their sequences. In both cases, we could generate edited MSTN equine cloned blastocysts: 3/153 (2%), 3/155 (2%), 8/140 (6%), and 9/73 (12%) for gRNA2-1μg-C2, gRNA2-5μg-C13, control fibroblasts, and control mesenchymal cells, respectively. In conclusion, genome edition by CRISPR/Cas9 is an efficient method to edit the genome of horse fibroblasts in a dose-dependent manner with apparent high specificity. Moreover, equine embryos can be generated with these cells with lower blastocyst rates than control fibroblasts of the same cell line or mesenchymal cells, probably due to higher cell passages needed for cell clone isolation and expansion. To the best of our knowledge, this is the first report of genome edited horse embryos.

Sensitive light-sheet microscopy in multiwell plates using an AFM cantilever.

We present a sensitive inverted light sheet microscope, capable of single-molecule fluorescence imaging of cells in 96-well plates. Light sheet microscope designs are often complex and costly, requiring custom-made sample chambers that are incompatible with standard cell culture samples. To overcome this limitation, we have developed single-objective cantilever selective plane illumination microscopy (socSPIM), which introduces a light sheet through the objective lens of an inverted microscope using an AFM tip. We demonstrate the effectiveness of this setup by performing 3D imaging of nuclear pore complexes, as well as live whole-cell 3D imaging of lysosomes and super-resolution imaging of the T-cell membrane. The unique advantage offered by socSPIM is the minimal footprint of the cantilever, which allowed us to perform super-resolution reflected light-sheet microscopy by PAINT in 96-well plates, paving the way for high-throughput studies.

An in vitro Study of Cytotoxic Activity of Euphorbia macroclada boiss on Mcf–7 Cells

Abstract: Objective: The study was aimed to evaluate the cytotoxic activity of acetone extract of leaves, flower and body of Euphorbia macroclada Boiss on human breast cancer cell line (MCF-7). Material: The cells were plated at a cell density of 1x105 cells in 96-well plates and grown with DMEM medium containing supplemented with 10% FBS and 1% penicillin. The cells were treated by different concentrations of acetone extract of Euphorbia macroclada Boiss (10–1000 μg/mL) during 24, 48 and 72 h. The cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard (3-(4,5- dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium bromide (MTT) assay. Results: After the evaluation of cytotoxicity assay results, it is determined that flower and body parts have a significant cytotoxic effect on MCF–7 breast cancer cell line. The values that obtained reading at 570 nm spectrophotometrically, were analyzed with GraphPad Prism7 and IC50 growth inhibition values was determined. Conclusion: The results of MTT assay showed that leaves, flower and body significantly reduced % cell viability comparative to the control. It was also shown that body had more growth inhibitory effect on MCF-7 cell compared to the leaves part. Key words: Euphorbia macroclada boiss, Cytotoxic activity, MCF-7, Breast cancer.

PO-391 High-throughput single-cell T-cell receptor profiling by SMART technology

IntroductionSingle-cell T-cell receptor (scTCR) clonotype analysis permits the determination of the specific TCR alpha-beta (α/β) chain pairing expressed on each cell. This pairing information allows researchers to gain insight into Tcell heterogeneity and plasticity, determine the contribution of the pairing to antigen specificity of the individual TCR, and design therapeutic antibodies. Here we employ a 5’-RACE-like approach and SMART technology, in conjunction with two novel next-generation sequencing (NGS) library preparation kits, using the same primer pairs, to capture full-length variable regions of TCR-α and -β chains.Material and methodsMethod 1, using the SMARTer Human scTCRa/b Profiling Kit, permits NGS library preparation of FACS-sorted cells in 96-well plates. Method 2 is an adaptation of the process above that scales to ~1200 single cells using the SMARTer ICELL8 Single-Cell System, which enables single-cell isolation and nanoliter PCR in a nanowell chip.Results and discussionsWe present data showing α/β pairing from Jurkat, CCRF, PBMCs, and CD4 +T cells. In addition to the sensitivity of this method, the ability to pool the cDNA from 96wells into 12 sequencing libraries adds to the ease of use. Consistent with immunology reports, unstimulated CCRF-CEM cells examined with this kit expressed a TCR-β but not a TCR-α chain.scTCR pofiling with SMARTer ICELL8 Single-Cell System as a proof of principle of scTCRα/β was performed on Jurkat cells and CCRF-CEM cells.Jurkat cells and CCRF-CEM cells were processed using an ICELL8 chip preprinted with barcoded oligos. Paired TCRα/β Jurkat clonotypes were detected in 77% and 87% cells in mixed and single cell populations, respectively.ConclusionThe ability of the core biochemistry and PCR components of these kits to be used with either FACS-sorted cells in 96-well plates or >1,000 cells in novel ICELL8 chips (in development) points to the general utility and scalability of this approach in understanding paired scTCR clonotype diversity.