Hts 7000

Manufactured by PerkinElmer

Sourced in United States

The HTS 7000 is a high-throughput screening (HTS) system designed for automated cell-based and biochemical assays. It features a 7-inch touchscreen interface, a 96- or 384-well microplate capability, and a temperature-controlled incubator. The system is capable of performing liquid handling, mixing, and detection tasks to support a variety of HTS applications.

Automatically generated - may contain errors

Lab products found in correlation

Dcfh da, by Olympus (3 mentions) Dcfh da, by Beyotime (3 mentions) Mineral oil, by Merck Group (2 mentions) Peg 4mal, by Laysan Bio (2 mentions) Biotin peg thiol, by Nanocs (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Dithiothreitol (dtt), by Merck Group (2 mentions) Span 80, by Merck Group (2 mentions) Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions) Dcfh da, by Merck Group (2 mentions) 96 well plate, by Corning (2 mentions) Dcfh da, by PerkinElmer (1 mentions) N dodecylphosphocholine, by Anatrace (1 mentions) Egg phosphatidylcholine, by Avanti Polar Lipids (1 mentions) Mitosox red, by Thermo Fisher Scientific (1 mentions) Molecular probes dcfh da, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions)

19 protocols using hts 7000

Measuring Oxidative Stress in Neuronal Cells

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The SH-SY5Y (1 × 10⁴ cells/well) and HT22 (1 × 10⁴ cells/well) cells were seeded in a 96-well cell culture plate for 24 h, and the primary hippocampal neurons (2 × 10⁵ cells/well) were seeded in a 24-well cell culture plate for 7 days. All cells were exposed to sevoflurane at concentrations of 4 and 8% for 12 h in the presence or absence of NAC. The levels of ROS were evaluated by redox-sensitive dye dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime Biotech, Nanjing, China) as we previously described (Wang et al., 2018 ). Cells were stained with 20 μmol/L DCFH-DA in a dark room for 30 min and the fluorescence density of DCFH-DA was calculated by a fluorescence spectrometer (HTS 7000, Perkin Elmer, Boston, MA, United States).
The levels of ROS in the hippocampi of rat pups were measured using an ROS ELISA detection kit (Jianglai Biotech, Shanghai, China) according to the manufacturer’s instructions. The extracted supernatant of hippocampus tissue samples was added to the ELISA scale, reacted with conjugate for 1 h at 37°C, and then reacted with substrate for 20 min at 25°C followed by stop solution. Then, the fluorescence absorptions were measured by a microplate reader (HTS 7000, Perkin Elmer, Boston, MA, United States) at 450 nm immediately. The concentration of ROS was calculated corresponding to the mean absorbance from the standard curve.

Piao M., Wang Y., Liu N., Wang X., Chen R., Qin J., Ge P, & Feng C. (2020). Sevoflurane Exposure Induces Neuronal Cell Parthanatos Initiated by DNA Damage in the Developing Brain via an Increase of Intracellular Reactive Oxygen Species. Frontiers in Cellular Neuroscience, 14, 583782.

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PEG-based Microgel Synthesis with Biotin

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A microgel precursor solution containing 5% w/v PEG-4MAL (20kDa, Laysan Bio) and 1.0 mM biotin-PEG-thiol (1 kDa, Nanocs) was reacted for 15 min in PBS. This precursor was dispersed into droplets and subsequently crosslinked within mineral oil (Sigma) containing 2% SPAN80 (Sigma) and a 1:15 emulsion of 30 mg/mL dithiothreitol (Sigma) on a microfluidic chip, as described previously ^{23 (link)}. Control microgels that did not contain biotin-PEG-thiol were also synthesized. After washing microgels 5 times by centrifugation in 1% bovine serum albumin (Sigma) in PBS, 10⁴ microgels were incubated with varying concentrations of a streptavidin-AlexaFluor488 conjugate for 30 min in 500 µL PBS, and were washed 5 times by centrifugation to remove unbound SA. Microgels from each sample were placed in a 96-well plate and fluorescence was measured on a plate reader (Perkin Elmer HTS 7000). Biotin and control microgels were also synthesized with a covalently bound peptide (GRGDSPC)-AlexaFluor594 conjugate for microgel visualization, and were fluorescently imaged to confirm biotin-specific SA immobilization.

Headen D.M., Woodward K.B., Coronel M.M., Shrestha P., Weaver J.D., Zhao H., Tan M., Hunckler M.D., Bowen W.S., Johnson C.T., Shea L., Yolcu E.S., García A.J, & Shirwan H. (2018). Local immunomodulation with Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance. Nature materials, 17(8), 732-739.

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Antimalarial Compound Screening Assay

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Ten microliters
of 10× compound diluted in RPMI 1640 media (Gibco) with a constant
concentration of 1% DMSO was added to a 96 well plate (Costar), 90
μL of 1.5% ring stage, synchronized with 5% w/v sorbitol P. falciparum 3D7 parasites, 1% hematocrit, in culture media
with 10% v/v human serum with 10 μg/mL gentamycin. Each compound
concentration and 1% v/v DMSO controls were run in triplicate. Plates
were incubated at 37 °C in 5% O₂, 5% CO₂, and 90% N₂ for 72 h. Plates were frozen, thawed, and
incubated with 100 μL 2× SYBR green in lysis buffer (20
mM Tris pH 7.5, 5 mM EDTA, 0.008% Saponin, 0.08% TritonX-100) in the
dark for at least 1 h. Fluorescence was measured with a plate reader
(HTS 7000, PerkinElmer) at excitation/emission wavelengths of 485/535
nm.

Hain A.U., Bartee D., Sanders N.G., Miller A.S., Sullivan D.J., Levitskaya J., Meyers C.F, & Bosch J. (2014). Identification of an Atg8-Atg3 Protein–Protein Interaction Inhibitor from the Medicines for Malaria Venture Malaria Box Active in Blood and Liver Stage Plasmodium falciparum Parasites. Journal of Medicinal Chemistry, 57(11), 4521-4531.

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Measuring Cellular Oxidative Stress

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Fluorescence probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA) was employed to evaluate ROS level. In short, 10 μΜ DCFH-DA (Sigma-Aldrich) was used to treat BRL-3A cells at 37°C for half an hour in the dark and then washed with serum-free DMEM for three times to fully remove the DCFH-DA that has not entered the cells. Finally, a fluorescence spectrometer (HTS7000, Perkin Elmer, USA) was applied to track ROS staining.

Zheng Y., Tao Y., Zhan X, & Wu Q. (2022). Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling. Bioengineered, 13(4), 8349-8359.

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Oxidative Stress Response in SH-SY5Y Cells

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SH-SY5Y cells (4×10⁴) were seeded onto 96-well microplate and cultured 24 h, and then treated with H₂O₂ at indicated concentrations. The average level of intracellular ROS was evaluated by using redox-sensitive dye DCFH-DA (Beyotime Biotech, Nanjing, China). All the experimental cells were washed twice in PBS and stained in the dark for 30 min with 20μmol/L DCFH-DA. After the cells were dissolved with 1% Triton X-100, the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength 530 nm using a fluorescence spectrometer (HTS 7000, Perkin Elmer, Boston, MA). The ROS levels were expressed as arbitrary unit/mg protein, then as the folds of control.
Other groups of SH-SY5Y cells were seeded onto a culture dish in a diameter of 3 cm and cultured 24 h. After being treated with H₂O₂, the cells were stained with DCFH-DA as described above, and observed under fluorescence microscope (Olympus IX71, Tokyo, Japan).

Gao Z., Wang H., Zhang B., Wu X., Zhang Y., Ge P., Chi G, & Liang J. (2018). Trehalose inhibits H2O2-induced autophagic death in dopaminergic SH-SY5Y cells via mitigation of ROS-dependent endoplasmic reticulum stress and AMPK activation. International Journal of Medical Sciences, 15(10), 1014-1024.

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Intracellular ROS Detection in PC12 Cells

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To detect intracellular ROS, PC12 cells were plated into 24-well plates. PC12 cells were stained with 10 μΜ 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich) at 37°C for half an hour in the dark and washed with serum-free RPMI 1640 medium three times. Finally, ROS staining was observed with the adoption of a fluorescence spectrometer (HTS7000, Perkin Elmer, USA).

Lin M., Li P., Liu W., Niu T, & Huang L. (2022). Germacrone alleviates okadaic acid-induced neurotoxicity in PC12 cells via M1 muscarinic receptor-mediated Galphaq (Gq)/phospholipase C beta (PLCβ)/ protein kinase C (PKC) signaling. Bioengineered, 13(3), 4898-4910.

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Erythrocyte Hemolysis by Bacterial Biofilm

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Human erythrocytes were washed with RPMI 1640 medium until the supernatant was visually free of hemoglobin pigment. The washed erythrocytes were suspended in malaria complete medium to yield a 1% hematocrit. Filtered Csp_P biofilm was mixed with erythrocytes and incubated up to 24 h at 37°C. To separate lysed RBC cytosol from whole RBCs, the suspension was centrifuged at 2000 rpm for 5 min. The resulting supernatant was carefully aspirated and plated in new 96-well microplates. Control erythrocytes without any bacterial material were used as a negative control (blank), and freeze-thawed erythrocyte lysate was used as positive control (100% hemolysate). To determine the % lysis in test samples, plates were read at 405 nm in an ELISA plate reader (HTS 7000 Perkin Elmer), and the reading was expressed as a fraction of the positive control.

Ramirez J.L., Short S.M., Bahia A.C., Saraiva R.G., Dong Y., Kang S., Tripathi A., Mlambo G, & Dimopoulos G. (2014). Chromobacterium Csp_P Reduces Malaria and Dengue Infection in Vector Mosquitoes and Has Entomopathogenic and In Vitro Anti-pathogen Activities. PLoS Pathogens, 10(10), e1004398.

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Evaluating Cell Viability in Neuronal Cells

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Reconstitution and Swelling Assay of OprG Protein

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The assay was performed as described previously (Nikaido and Rosenberg, 1983 (link)) with the following modifications. Liposomes were made by suspending a dried film of 3 μmoles egg phosphatidylcholine (Avanti Polar Lipids, Alabaster, Al) and 0.15 μmoles egg L-α-phosphatidic acid (Avanti Polar Lipids, Alabaster, Al) in 20 μL of a 1.875 mM micellar solution of n-dodecylphosphocholine (Anatrace, Maumee, OH) in water containing 2 (protein-to-lipid ratio 1:1575) or 6 (protein-to-lipid ratio 1:525) nmoles OprG, sonicated for 15 min, and completely dehydrated in vacuo. After rehydration in assay buffer (12 mM stachyose, 10 mM HEPES-NaOH, pH 7.5) under vigorous shaking and 2 times 15 min sonication, a suspension of multilamellar vesicles containing OprG in their membranes was obtained. Reconstitution of OprG was quantitative as far as detectable on Coomassie stained SDS-PAGE gels (data now shown).
In a 96 well plate (Corning #3631), 5 μL OprG liposomes were diluted with 200 μL of an equiosmolar analyte solution in 10 mM HEPES-NaOH, pH 7.5, and absorbance was monitored at 405 nm using a Perkin-Elmer HTS 7000 plate reader over a period of 5 min. Data was analyzed following published procedures (Bangham et al., 1967 ; Nikaido and Rosenberg, 1983 (link)) and swelling rates were reported relative to L-alanine.

Kucharska I., Seelheim P., Edrington T., Liang B, & Tamm L.K. (2015). OprG harnesses the dynamics of its extracellular loops to transport small amino acids across the outer membrane of Pseudomonas aeruginosa. Structure (London, England : 1993), 23(12), 2234-2245.

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Intracellular and Mitochondrial ROS Assay

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Intracellular ROS was evaluated by using DCFH-DA (Beyotime Biotech, Nanjing, China) according to manufacturer’s instruction. The fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength 530 nm using a fluorescence spectrometer (HTS 7000, Perkin Elmer, Boston, MA). The ROS levels were expressed as arbitrary unit/mg protein, then as the percentage of control.
Mitochondrial superoxide was assayed by using MitoSOX red according to manufacturer’s description (Invitrogen, Eugene, OR). The red fluorescence density was measured at an excitation wavelength of 510 nm and an emission wavelength at 580 nm, and was expressed as a ratio to the fluorescence in control cells.
Other groups of cells were seeded onto a 6-well culture plate, stained with DCFH-DA or MitoSOX red as described above, and observed under fluorescence microscope (Olympus IX71, Tokyo, Japan).

Wang X., Fan L., Wang X., Luo T, & Liu L. (2022). Cyclophilin A contributes to shikonin-induced glioma cell necroptosis and promotion of chromatinolysis. Scientific Reports, 12, 14675.

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