Arrayscan vti hcs, by Thermo Fisher Scientific - Product details

1

Neurotoxicity and Peripheral Toxicity Assays

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For the NeuriTox test, LUHMES cells were differentiated for 48 h and seeded into 96 well plates at a density of 100,000 cells/cm² in a volume of 90 μl DM without cAMP and GDNF. Treatment was initiated applying 10 μl of a 10x concentrated treatment solution one hour after seeding. At 24 h after treatment, staining mix (SM) was applied (final concentrations: 1 μg/ml H-33342, 1 μM calcein-AM).
For the PeriTox test method, the cells were thawed and seeded at a density of 100,000 cells/cm² in 75 μl PeriTox differentiation medium (PDM) consisting of 25% KSR-S and 75% N2-S media supplemented with 1.5 μM CHIR99021, 1.5 μM SU5402, and 5 μM DAPT on matrigel-coated plates (KSR-S: knockout DMEM with 15% serum replacement, 1 x Glutamax, 1 x nonessential amino acids and 50 mM beta-mercaptoethanol; N2-S: DMEM/F12, with 2 mM Glutamax, 0.1 mg/ml apotransferrin, 1.55 mg/ml glucose, 25 μg/ml insulin, 100 mM putrescine, 30 nM selenium, and 20 nM progesterone). After one hour, 25 μl PDM with 4x concentrated serial dilutions of the test compounds was added to the cells. At 23 h after treatment, the cells were stained with SM and incubated for one additional hour at 37°C.
Image acquisition was performed with an ArrayScan VTI HCS (high content imaging) microscope (Cellomics, Waltham, MA, USA).

Delp J., Gutbier S., Klima S., Hoelting L., Pinto-Gil K., Hsieh J.H., Aichem M., Klein K., Schreiber F., Tice R.R., Pastor M., Behl M, & Leist M. (2018). A High-Throughput Approach to Identify Specific Neurotoxicants / Developmental Toxicants in Human Neuronal Cell Function Assays. ALTEX, 35(2), 235-253.

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Neurotoxicity and Peripheral Toxicity Assays

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For the NeuriTox test, LUHMES cells were differentiated for 48 h and seeded into 96 well plates at a density of 100,000 cells/cm² in a volume of 90 μl DM without cAMP and GDNF. Treatment was initiated applying 10 μl of a 10x concentrated treatment solution one hour after seeding. At 24 h after treatment, staining mix (SM) was applied (final concentrations: 1 μg/ml H-33342, 1 μM calcein-AM).
For the PeriTox test method, the cells were thawed and seeded at a density of 100,000 cells/cm² in 75 μl PeriTox differentiation medium (PDM) consisting of 25% KSR-S and 75% N2-S media supplemented with 1.5 μM CHIR99021, 1.5 μM SU5402, and 5 μM DAPT on matrigel-coated plates (KSR-S: knockout DMEM with 15% serum replacement, 1 x Glutamax, 1 x nonessential amino acids and 50 mM beta-mercaptoethanol; N2-S: DMEM/F12, with 2 mM Glutamax, 0.1 mg/ml apotransferrin, 1.55 mg/ml glucose, 25 μg/ml insulin, 100 mM putrescine, 30 nM selenium, and 20 nM progesterone). After one hour, 25 μl PDM with 4x concentrated serial dilutions of the test compounds was added to the cells. At 23 h after treatment, the cells were stained with SM and incubated for one additional hour at 37°C.
Image acquisition was performed with an ArrayScan VTI HCS (high content imaging) microscope (Cellomics, Waltham, MA, USA).

Delp J., Gutbier S., Klima S., Hoelting L., Pinto-Gil K., Hsieh J.H., Aichem M., Klein K., Schreiber F., Tice R.R., Pastor M., Behl M, & Leist M. (2018). A High-Throughput Approach to Identify Specific Neurotoxicants / Developmental Toxicants in Human Neuronal Cell Function Assays. ALTEX, 35(2), 235-253.

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3

Immunofluorescence Analysis of Cellular Markers

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The expression of CD31, α-SMA, HO-1 and nuclear translocation of Nrf2 were determined using immunofluorescence staining. After treatment, cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde. Then, cells were permeabilized with 0.5% Triton X-100 and blocked for 1 hours in 3% BSA at 37°C. Cells were immunostained with primary antibodies overnight at 4 °C, and then incubated with Alexa 488 or Alexa 555-conjugated secondary antibody (diluted 1:500) for 1 hour at room temperature. Nuclei were dyed with Hoechst 33342. Images were obtained and analyzed with a HCS Kinetic Scan (Cellomics ArrayScan VTI HCS) immediately.

Chen Y., Yuan T., Zhang H., Yan Y., Wang D., Fang L., Lu Y, & Du G. (2017). Activation of Nrf2 Attenuates Pulmonary Vascular Remodeling via Inhibiting Endothelial-to-Mesenchymal Transition: an Insight from a Plant Polyphenol. International Journal of Biological Sciences, 13(8), 1067-1081.

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Intracellular ROS Generation Assay

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The intracellular generation of ROS, as an index of oxidative stress, was determined using DCFH-DA. DCFH-DA is a cell-permeable probe. After diffusing into cells, it could be metabolized into the nonfluorescent DCFH, which could be oxidized by intracellular ROS into highly fluorescent DCF. After incubation with doses of SAA in the presence or absence of TGFβ1, HPAECs were rinsed with PBS and incubated with 5 µM DCFH-DA in serum-free medium in a light-free chamber for 30 min. Nuclei were dyed with Hoechst 33342. Images were obtained and analyzed with a HCS Kinetic Scan (Cellomics ArrayScan VTI HCS).

Chen Y., Yuan T., Zhang H., Yan Y., Wang D., Fang L., Lu Y, & Du G. (2017). Activation of Nrf2 Attenuates Pulmonary Vascular Remodeling via Inhibiting Endothelial-to-Mesenchymal Transition: an Insight from a Plant Polyphenol. International Journal of Biological Sciences, 13(8), 1067-1081.

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5

Evaluating PCV2 Variant Binding Reactivity

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PK15 cells were infected with the same titer of PCV2a, 2b, 2d-1 and 2d-2 as 200 TCID₅₀. For evaluating the binding reactivity of PCV2 and mAbs, infected PK15 cells were fixed with 80% acetone for 10 min at − 20 °C. After washing with 1 × PBS, mAbs (1:200) as primary antibodies were reacted by incubating for 1 h at room temperature (RT). After rinsing, Alexa fluor™ 488 goat anti-mouse immunoglobulin G (IgG, 1:200, Invitrogen, CA, USA) as a secondary antibody was incubated for 30 min at RT. The nuclei were stained for 5 min with Hoechst33258 (Invitrogen) diluted in 1 × PBS (1:10,000). For the detection of PCV2-infected cells, fluorescent intensity of PCV2-positive cells per 1 × 10⁴ cellular nuclei was measured by ArrayScan VTI HCS (Thermo Scientific, MA, USA), and the number of PCV2-positive cells was counted with by the naked eye.

Kang S.J., Kang H., You S.H., Lee H.J., Lee N., Hyun B.H, & Cha S.H. (2020). Genetic diversity and different cross-neutralization capability of porcine circovirus type 2 isolates recently circulating in South Korea. BMC Veterinary Research, 16, 334.

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6

High-Content Screening of Mitotic Cells

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Cells were plated at 10 000/well in 96-well plates and incubated overnight. The following day compound stocks in DMSO were diluted in medium then added to cells with a maximum final DMSO concentration on cells of 0.2%. Cells were incubated with compound for 24 h then fixed in 3.7% formaldeyde. Cells were permeabilised with 0.1% Triton X-100 then incubated with anti-phospho-histone H3 (Ser10) antibody (Abcam ab5176). The cells were washed with PBS then incubated with AlexaFluor 488 labelled goat anti-rabbit IgG (Invitrogen A11034) in the presence of 4ug/ml Hoechst 33342 (Invitrogen H3570). Cells were washed in PBS then imaged on an Arrayscan VTi HCS instrument (Thermo Fisher) using the Target Activation V4 Bioapplication. A user-defined threshold was applied to identify mitotic cells based on the intensity of phospho-histone H3 staining.

Narvaez A.J., Ber S., Crooks A., Emery A., Hardwick B., Guarino Almeida E., Huggins D.J., Perera D., Roberts-Thomson M., Azzarelli R., Hood F.E., Prior I.A., Walker D.W., Boyce R., Boyle R.G., Barker S.P., Torrance C.J., McKenzie G.J, & Venkitaraman A.R. (2017). Modulating Protein-Protein Interactions of the Mitotic Polo-like Kinases to Target Mutant KRAS. Cell Chemical Biology, 24(8), 1017-1028.e7.

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7

Quantifying Apoptosis in Cell Cultures

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Cells were inoculated in a 96-well plate and grown to 75% confluence. Cells in the logarithmic growth phase were collected. After washing with RPMI-1640 medium without serum, cells were incubated in 100 μl RPMI-1640 medium without serum [50 mg/ml propidium iodide (Sigma-Aldrich, St Louis, Missouri, USA), 5 μg/ml Hoechest 33342 (Thermo, Hom Bridge City, Massachusetts, USA)]. Cells were incubated for 10 m at 37°C and the percentage of apoptotic cells was determined by Thermo Scientific ArrayScan VTI HCS (Thermo). Cellquest software (Becton Dickinson, Franklin, New Jersey, USA) was used for analysis. This experiment was repeated three times.

Wang H., Li W., Lai B., Yang X., Zhang C., Li J, & Zhu Y. (2015). A better experimental method to detect the sensitivity of cancer cells to anticancer drugs after adenovirus-mediated introduction of two kinds of p53 in vivo. Anti-Cancer Drugs, 26(8), 852-859.

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8

Chlamydia Trachomatis Reinfection Assay

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Reinfection experiments were performed as previously described.10 (link)C. trachomatis serovar L2 EBs were mixed with Hank balanced salt solution (HBSS) and used to infect HeLa cells at a MOI of 0.3–0.5. After 1 h, the compounds were added mixed with RPMI at 2.5 μM or 1 μM. The cells were incubated for 44–48 h, then cold Milli-Q water was added in order to osmotically lyse the cells and harvest the EBs. 4× SPG was added to the EBs on MQ water to reach 1× SPG, and 10-fold serial dilutions were performed in HBSS. Fresh HeLa cells were infected with the diluted EBs, substituting the HBSS by fresh RPMI medium 1 h post infection. Cells were incubated for 44–46 h before fixation with methanol. Inclusions were stained with in-house rabbit anti-Chlamydia antibodies21 (link) (1 : 1000), followed by secondary donkey anti-rabbit FITC-labeled antibody (1 : 1000) (Jackson ImmunoResearch). Cell nuclei were stained with DAPI (1 : 1000). Infected cells were observed and measured by Arrayscan automated microscopy (ArrayScan VTI HCS, Thermo Scientific). Reinfect value was calculated as the percentage of IFUs compared to a DMSO treated control. The presented value is the average of three independent experiments with duplicates for each condition.

Kulén M., Núñez-Otero C., Cairns A.G., Silver J., Lindgren A.E., Wede E., Singh P., Vielfort K., Bahnan W., Good J.A., Svensson R., Bergström S., Gylfe Å, & Almqvist F. (2019). Methyl sulfonamide substituents improve the pharmacokinetic properties of bicyclic 2-pyridone based Chlamydia trachomatis inhibitors. MedChemComm, 10(11), 1966-1987.

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9

High-Throughput Anticancer Compound Screening

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S17 cells (1.0 × 10³ /well) and PhLO cells (1.0 × 10⁴ /well) were seeded on 96-well plates in 10% FBS-containing RPMI medium on day 1. On day 2, the library compounds (2 μM each) were added to each well. After 48 hours (h) on day 4, total and dead PDX cells were stained with Hoechst 33342 and Propidium Iodide (PI), respectively. Total and dead PDX cells were counted separately from S17 cells using an Array Scan VTI HCS image analyzer (Thermo Fisher Scientific, Waltham, MA, USA) as described previously. [15 (link)] Since the library contained some photosensitive compounds, all experiments were performed under conditions that avoided sunlight and laser light.

Morishita T., Hayakawa F., Sugimoto K., Iwase M., Yamamoto H., Hirano D., Kojima Y., Imoto N., Naoe T, & Kiyoi H. (2016). The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with dasatinib. Oncotarget, 7(35), 56241-56252.

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Arrayscan vti hcs

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9 protocols using arrayscan vti hcs

Neurotoxicity and Peripheral Toxicity Assays

Neurotoxicity and Peripheral Toxicity Assays

Immunofluorescence Analysis of Cellular Markers

Intracellular ROS Generation Assay

Evaluating PCV2 Variant Binding Reactivity

High-Content Screening of Mitotic Cells

Quantifying Apoptosis in Cell Cultures

Chlamydia Trachomatis Reinfection Assay

High-Throughput Anticancer Compound Screening

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