The extraction process was based on the method described by Bligh and Dyer [14 (link)]. Briefly, after extensive wash, the cells were pelleted and resuspended in a lysis buffer (Beyotime, China) containing 1 mM phenylmethanesulfonylfluoride (PMSF). The supernatant was collected after centrifugation at 12000 rpm for 15 min at 4 °C. Protein concentrations were measured using the Enchanced BCA Protein Assay Kit (Beyotime, China). 100 μl of lysate was placed in a microtube, and then 125 μl of chloroform and 250 μl of methanol were added. The mixture was vortexed for 1 min. The apolar and polar phases were divided by the addition of 125 μl of chloroform and 125 μl of distilled water. The mixture was centrifuged at 1000 rpm at room temperature for 5 min. The lower lipid containing phase was recovered and transferred to a clean microtube. Total cholesterol (TC) and free cholesterol (FC) contents were determined using the Cholesterol Quantification Kit (Sigma Aldrich, USA) and measured using a BioTek Synergy NEO (BioTek Instrument, USA) at the absorbance of 570 nm. CE was determined as the difference between TC and FC (TC minus FC) and was expressed in units of mg/g cellular protein.
Biotek synergy neo
Manufactured by Agilent Technologies
Sourced in United States
The BioTek Synergy NEO is a multi-mode microplate reader designed for a variety of applications in life science research. It features a modular design, allowing for the incorporation of different detection modes, including absorbance, fluorescence, and luminescence. The Synergy NEO provides high-performance detection capabilities and can be customized to meet the specific needs of various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
One glo ex luciferase assay system, by Promega (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Cholesterol quantification kit, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Neon transfection system starter pack, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Hoechst, by Agilent Technologies (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Hoechst, by Beyotime (1 mentions)
880 airyscan, by Zeiss (1 mentions)
Jc 1 kit, by Beyotime (1 mentions)
Polystyrene microplates, by Corning (1 mentions)
Prism version 8, by GraphPad (1 mentions)
Hek293 cells, by Corning (1 mentions)
Prism v8, by GraphPad (1 mentions)
Adp glo reagent, by Promega (1 mentions)
Kinase detection reagent, by Promega (1 mentions)
Nebuffer 2, by New England Biolabs (1 mentions)
Neo lite luciferase assay system, by PerkinElmer (1 mentions)
Envision multimode plate reader, by PerkinElmer (1 mentions)
9 protocols using biotek synergy neo
1
Cholesterol Quantification in Cells
2
Myogenic Transcription Factor Regulation
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MyoG promoter (+3 ~ −1596), MyoG promoter + E1 (WT or Mutation), and MyoG promoter + E2 (WT or Mutation) were cloned into pGL3 basic vector and transfected to primary myoblasts using the Neon™ Transfection System Starter Pack (Invitrogen, MPK5000S), respectively. After transfection 48 h, luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega, PR-E1910) by BioTek Synergy NEO (BioTek) following the manufacturer’s instructions. Relative luciferase activity was calculated as the ratio of Firefly/Renilla luciferase activity. All experiments were repeated at least 3 times. Primers were listed in Tables S3 and S4 .
3
Quantifying Cellular Oxidative Stress
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Intracellular ROS production was detected using the ROS Assay Kit (Beyotime, S0033). Briefly, cells were initially seeded in a 96-well plate at a density of 1 × 104 cells/well and subsequently treated with or without 10 μM Aβ. After completion of the treatment, the cells were co-stained with 10 μM DCFH-DA and 3 μg/mL Hoechst (Beyotime, C1022) at 37 °C for 20 min. Then, cells were washed twice with PBS, and the ROS levels were determined using BioTek SynergyNEO (BioTek, Winusky, VT, USA) at excitation/emission wavelengths of 488/525 nm for DCFH-DA and 350/461 nm for Hoechst. Alternatively, the cells in the 96-well black plate were observed using a laser-scanning confocal microscope (Operetta, Perkin Eimer, Waltham, MA, USA).
4
Mitochondrial Membrane Potential Assay
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HMC3 cells were plated at 9,000 cells per well in black-walled 96-wells plates and cultured overnight, then stimulated with 10 μM Aβ for 72 h. JC-1 kit (Beyotime, #C2006) was used to detect the MMP level of cells according to the manufacturer's instructions. In brief, cells were loaded with JC-1 staining solution for 30 min at 37°C and then washed with staining buffer for twice. The cells were captured using Zeiss confocal laser scanning microscope (Zeiss 880 Airyscan). The fluorescence intensity was measured at 490/530 nm (green) for monomers and 525/590 nm for aggregates (red) using the BioTek SynergyNEO (BioTek), and the ratio of red/green fluorescence intensity was presented as MMP.
5
ACE2-Mediated SARS-CoV-2 Pseudovirus Entry Inhibition
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HEK 293 cells (ATCC® CRL-1573) stably over-expressing full-length human ACE2 protein were seeded in 96-well white polystyrene microplates (Corning, Cat. No. CLS3610) at 0.03 × 106 cells/well in DMEM (10% FBS and 1% Pen-Strep), and grown overnight at 37 °C, 5% CO2. To test the inhibition of pseudovirus entry by ACE2 WT or mutants, increasing concentration of Fc-tagged ACE2 proteins were first mixed with pseudoviruses and incubated at room temperature for 10 m72 . The ACE2 WT or mutant treated pseudovirus mixture was then used to infect cells. The cells were incubated at 37 °C, 5% CO2 for 6 h, then the medium was replaced with fresh DMEM (10% FBS and 1% Pen-Strep), and then again every 24 h for up to 72 h. To measure the luciferase signal (a proxy for virus uptake), DMEM was removed and cells were replaced in DPBS (ThermoFisher, Cat. No. 14190250) and mixed with an equal volume of ONE-Glo™ EX Luciferase Assay System (Promega, Cat. No. 8130). Relative luciferase units were measured using a BioTek Synergy Neo plate reader (BioTek Instruments Inc.). The data was then analyzed using GraphPad Prism Version 8.4.3 (GraphPad Software, LLC.).
6
Evaluating AURKA ATPase Activity
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The ATPase activity of recombinant AURKA was evaluated using the ADP-Glo assay Kinase kit (Promega). Bora1–224 or pBora1–224 was titrated into 10 nM of AURKAWT or AURKAT288V in a buffer containing 40 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, and 0.01% Brij 35. The final reaction volume was 20 µL. When 100 μM Kemptide (LRRASLG, Cedarlane) was present in the reaction mix, the AURKA concentration was reduced to 2 nM. Reactions were initiated by the addition of 10 μM of ATP and incubated at room temperature for 60 min. Reactions were terminated by transferring 10 µL of the reaction mix to a 384-well white plate (Lumitrec 200, VWR) and adding 10 µL of ADP-Glo™ Reagent (Promega). After a 40-min incubation, 20 µL of kinase detection reagent (Promega) was added and allowed to incubate for an additional 30 min. Luminescence was measured on a Biotek Synergy Neo plate reader (BioTek) using a 1 s integration time. Results were plotted in GraphPad Prism V8.4.2 using a one-site total binding curve fitting analysis.
7
Pseudotyped SARS-CoV-2 VLP Entry Assay
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HEK-293 cells stably over-expressing full-length human ACE2 protein were seeded in 96-well white polystyrene microplates (Corning, Cat. No. CLS3610) at 0.03 × 106 cells/well in DMEM (10% FBS and 1% penicillin–streptomycin) and were grown overnight at 37 °C with 5% CO2. Pseudotyped VLPs were mixed with Ab, incubated at room temperature for 10 min, and added to the cells. The cells were incubated at 37 °C with 5% CO2, the medium was replaced with fresh DMEM (10% FBS and 1% penicillin–streptomycin) after 6 h, and again every 24 h up to 72 h. To measure the luciferase signal (VLP entry), DMEM was removed and cells were replaced in DPBS (ThermoFisher) and mixed with an equal volume of ONE-Glo™ EX Luciferase Assay System (Promega). Relative luciferase units were measured using a BioTek Synergy Neo plate reader (BioTek Instruments Inc.). The data were analyzed by GraphPad Prism Version 8.4.3 (GraphPad Software, LLC).
8
Trans-cleavage Kinetic Characterization
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The trans-cleavage kinetic experiment was carried out following Cofsky et al.14 with modifications. In short, BrCas12b, sgRNA, and dsDNA activator were combined to a final concentration of 100 nM : 125 nM : 1 nM, respectively in 1x NEBuffer 2·1 (New England Biolabs) and incubated at 62 °C for 30 min. HEX-polyT-Quencher reporter (FQ) at various concentrations (10 nM, 100 nM, 200 nM, 500 nM, 1 µM, and 2 µM) was added to the reaction mixture containing Cas12b trans-activated complex; and the entire reaction was then immediately transferred to a plate reader. Fluorescence measurements (λex: 483/30 nm, λem: 530/30 nm) were read every 30 s using the Biotek Synergy Neo (Agilent) that was pre-heated to 62 °C. Initial velocity for each FQ concentration was determined by establishing the slope for all components and subtracting the slope for the no-activator control. The cleaved HEX-polyT reporter was titrated to different concentrations (10 nM, 100 nM, 200 nM, 500 nM, 1 µM, and 2 µM) and measured in the same experiment for the conversion of FQ fluorescence to concentration, eliminating non-linearity at high reporter concentrations.
9
Quantifying Ad26 Neutralizing Antibodies
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Ad26 Nab titers in serum were assessed using a luciferase-based virus neutralization assay (VNA)50 (link). Briefly, heat-inactivated Cynomolgus Macaque serum samples was 2-fold serial diluted starting at a 1:32, 1:64 or 1:128 dilution for Ad26 (depending on the study, see figure legends for the exact start dilution). E1/E3 deleted Ad26-luciferase reported constructs were combined with serial diluted serum into a Tissue Culture treated Black and White Isoplate-96 (Perkin Elmer, Nederland B.V) at 500 to 1000 vp/cell. Plates were incubated for 30 minutes at RT, before A549 human lung carcinoma cells (ATCC® CCL-185™, American Type Culture Collection, Manassas, VA, USA) were added at a density of 1×104 cells/well. After incubation for 20 to 24 hours at 37 °C and 10% CO2, luciferase activity was measured using the Neo-Lite Luciferase Assay System (Perkin Elmer, Waltham, MA, USA) and a BioTek Synergy Neo luminescence counter (BioTek/Agilent, Santa Clara, CA, USA) or EnVision multimode plate reader (Perkin Elmer). A 90% neutralization titer (IC90) was defined as the maximum serum dilution that neutralized 90% of luciferase activity. Each serum sample was analyzed in duplicate.
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