Streptavidin Coated pre-blocked plates (Thermofisher, 15124) were washed 3 times with 200 μL of phosphate buffered saline with 0.1% tween (PBS-T) prior to coating with 100 μL of biotinylated anti-ApoA-I antibody (abcam, ab27630) diluted 1:1400 in PBS-T. Plates were incubated at 37 °C for 2 hours and subsequently washed 6 times with PBS-T. Plasma samples diluted 1:200 in PBS with 0.05% casein (PBS-C) was added to the wells and incubated at 37 °C for 30 min. Following 6 washes with PBS-T, 100 μL of horseradish peroxidase (HRP)-conjugated anti-Human IgG (abcam, ab7153) was added at a dilution of 1:4000 in PBS-C and incubated for 30 min at 37 °C. After a final 6 washes with PBS-T, 100 μL of room temperature tetramethylbenzidine (TMB) (Rockland Immunochemical) was added and allowed to react for 30 min at room temperature in the dark. The reaction was stopped using 100 μL of 0.5 M H2SO4 and absorbance was measured at 450 nm (Biotek, Synergy Hybrid Reader). All samples were analyzed in duplicate.
Synergy hybrid reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy Hybrid Reader is a versatile instrument designed for a wide range of life science applications. It combines multiple detection technologies, including absorbance, fluorescence, and luminescence, in a single platform. The Synergy Hybrid Reader provides researchers with a powerful tool to perform a variety of assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Adcc reporter bioassay kit, by Promega (5 mentions)
Nt probnp, by Agilent Technologies (2 mentions)
Ck mb, by Agilent Technologies (2 mentions)
Ck mb, by Mlbio (2 mentions)
Nt probnp, by Mlbio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ab27630, by Abcam (1 mentions)
Ab7153, by Abcam (1 mentions)
Enliten atp assay system, by Promega (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
96 well plate, by Greiner (1 mentions)
Prism 5, by GraphPad (1 mentions)
Prism 7, by GraphPad (1 mentions)
Ht dna, by Thermo Fisher Scientific (1 mentions)
Ht dna, by Merck Group (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
19 protocols using synergy hybrid reader
1
Quantification of Plasma ApoA-I Antibodies
2
ADCC Receptor Engagement Assay
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Using the Promega ADCC reporter bioassay kit and a published protocol, in vitro engagement of the ADCC receptor was assessed [35 ]. MDCK cells were infected at a multiplicity of infection of 1 overnight with each respective virus, as mentioned earlier in the IF assay section. The next morning, antibody dilutions were added onto the cells in addition to 75,000 effector cells per well. Cells were then left in the 37°C incubator for six hours. The luciferase substrate was added in the dark and the luminescence activity was read after 5 min using Synergy Hybrid Reader (BioTek). Anti-stalk mAb CR9114, which has known ADCC reporter activity and binding to H4 HA, was used as a positive control [29 (link),57 (link),58 (link)]. Fold induction over an irrelevant antibody (anti-Lassa GPC) was calculated and data were analysed in Prism 7.
3
Liver Injury Assessment in Rats
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The rats were sacrificed after the last treatment. Blood samples were collected and centrifuged at 3000 × g for 10 min to obtain serum. All serum samples were sterile, hemolysis-free and stored at -70°C before determining the biochemical parameters and conducting the metabolomics analysis. The serum levels of ALT, AST, TBIL, DBIL, TBA, and ALP were measured on a Synergy hybrid reader (Biotek, Winooski, VT, USA). The livers were excised and fixed in 10% PBS-buffered formalin. Three or four paraffin-embedded sections (4–5 μm thick) per specimen were prepared and stained with hematoxylin-eosin (HE staining). The stained sections were examined under a Nikon microscope (Tokyo, Japan) and analyzed using the image Pro-Plus 7200 software.
4
Colorimetric Cell Growth Quantification
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Cell growth was monitored by using the diphenylamine colorimetric method41 (link). For growth quantification, cell pellets were harvested from 1 ml cultures by centrifugation at 10,000 × g at room temperature for 10 min and washed twice with the appropriate buffer. The cell pellets were then resuspended with 2 ml diphenylamine reagent and incubated at 60 °C for 1 h. After centrifugation, the supernatants were transferred to 96-well microtiter plate and measured at 59 nm by a multifunctional microtiter plate reader (Synergy Hybrid Reader, BioTek, USA).
5
Intracellular ATP Quantification in Mtb
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Intracellular ATP was quantified by using the bioluminescence based Enliten ATP assay system (Promega). Duplicate aliquots of Mtb mycobacteria (100 μl) were collected at various time points and immediately heat-inactivated. Cell lysates (25 μl) were transferred into 96-well white plates and the assay was performed as per manufacturer’s instructions. The emitted luminescence was detected with a Synergy Hybrid Reader (Biotek Instruments) and was expressed as relative luminescence units. ATP standards were included in all experiments as internal controls.
6
Serum Biomarkers and Cell Damage Assessment
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Serum biochemical indices, including BNP, NT-proBNP, LDH, CK-MB, and AST were determined on a Synergy hybrid reader (Biotek, Winooski, USA). In addition, the serum energy metabolism-related indices, including ATP, ATPase, NAD, NADH were also detected. BNP, NT-proBNP, LDH, and CK-MB were recruited from Shanghai MLBIO Biotechnology Co., Ltd. AST was obtained from Nanjing Jiancheng Bioengineering Institute. ATP, ATPase, NAD, and NADH were purchased from Shanghai Kanglang Biotech Co., Ltd. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to indicate the cytotoxicity, cell damage and its recovery.
7
ABTS+ Scavenging Ability of Nanoparticles
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The ABTS+ scavenging ability was determined as described in Budluang et al. [25 (link)]. Briefly, 7 mM ABTS+ stock solution was mixed with potassium 2.45 mM (1:1, v/v) persulfate and left in the dark at room temperature for 12–16 h until the reaction was complete and the absorbance was stable. The ABTS+ reagent was diluted with DI H2O to an absorbance of 0.700 ± 0.02 at 734 nm (Microplate reader Synergy Hybrid Reader, BioTek). The nanoparticle solution (100 µL) was mixed with the ABTS+ solution (100 µL) in multiple 96-well plates. Deionized H2O was used as a control. The absorbance was read at 734 nm after the reaction every 15 min until 60 min. The antioxidant activity was calculated as % inhibition using the following equation.
8
MTT Assay for Cell Viability
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The cells were seeded at 10,000 cells per well in a 96-well plates in normal growth medium. 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reagent was added to each well and incubated for an additional 4 h at 37°C with 5% CO2. Media was aspirated and the precipitate was solubilized in 100 μl 10%SDS. The absorbance of each well was measured at 547 nm by Synergy Hybrid Reader (BioTek Instruments, Inc) according to the manufacturer’s instructions. The percentage of viable cells was calculated relative to control wells.
9
CCK8 Assay for Cell Proliferation
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A Cell Counting Kit 8 (Dojindo, Kumamoto, Japan) was used to detect cell proliferation according to the user guidelines. Briefly, 1 × 103 HCECs were added to 96-well-plates in 100 µl cell culture medium per well. The cells were then treated with 500 ng/ml IFN-γ for 24 h or not treated at all. Next, CCK8 solution was injected into each well for 1 h at 37°C until the color changed. The absorbance of each sample was determined at 450 nm using an Epoch microplate spectrophotometer (Bio Tek, Biotek Winooski, VT, USA) and a Synergy hybrid reader (Bio Tek). The assay was repeated in triplicate.
10
ADCC Assay for Antibody Activity
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An ADCC reporter bioassay kit (Promega) was used to assess whether any of the MAbs elicit ADCC activity. The protocol has been described previously but was modified as needed (14 (link), 23 (link)). Vero.E6 cells (50,000 per well) were added onto white round-bottom 96-well cell culture plates (Corning Costar) and infected with VSV-LASV at a multiplicity of infection (MOI) of 1.0. Virus was prepared in MEM, and this medium was removed from the cells after 16 h. Twofold serial dilutions of each antibody were added to the cells in duplicate starting from a concentration of 100 µg/ml. Seventy-five thousand effector cells were added to the cells with the antibody dilutions, and cells were incubated at 37°C for 6 h. Luciferase substrate was added, and luminescence was measured 2 to 5 min later using a Synergy Hybrid Reader (BioTek). Human monoclonal antibody CR9114 was used on influenza virus (A/duck/Czechoslovakia/1956 H4N6)-infected Vero.E6 cells as a positive control (42 (link), 43 (link)).
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