ATP levels were determined by adaptation of the method of Patton et al. S. aureus RN450 cells were grown from an overnight culture to exponential phase (37°C, shaking at 200 rpm). Cultures were treated with 1 μg/mL daptomycin, 0.125 μg/mL nigericin, or EtOH for 1 h. After treatment, cells were concentrated by centrifugation and resuspended in PBS. ATP concentrations were determined using an ATP bioluminescent assay kit (Beyotime, cat.#S0026B, Shanghai, China) according to the manufacturer’s instructions. Bioluminescence was measured by a Thermo Fisher Scientific Varioskan Flash Multiplate Reader.
Varioskan flash multiplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Varioskan Flash Multiplate Reader is a versatile instrument designed for high-throughput microplate-based detection and analysis. It offers a range of detection modes, including absorbance, fluorescence, and luminescence, enabling a diverse set of applications in life science research and drug discovery.
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Lab products found in correlation
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Horseradish peroxidase, by Merck Group (2 mentions)
Luminol, by Merck Group (2 mentions)
Atp bioluminescent assay kit, by Beyotime (1 mentions)
96 well plate, by NEST Biotechnology (1 mentions)
Kgp902, by Keygen Biotech (1 mentions)
Elf18 peptide, by GenScript (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
16 protocols using varioskan flash multiplate reader
1
Measuring ATP Levels in S. aureus
2
Cytotoxicity Evaluation of Nanoparticles
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Cytotoxicity evaluation was conducted by MTT assay. Caco-2 cells were seeded in 96-well plates (NEST Biotechnology Co. Ltd., China) at a density of 5 × 103 cells/well and then cultured for 72 h. After the cells were exposed to NAR, NARNCs, and NAR Sol for 12 h, the medium was replaced with 200 µl of the MTT solution (0.5 mg/ml in PBS), and the cells were incubated for 4 h. The supernatant was then discarded, and dimethyl sulfoxide (200 µl/well) was added to dissolve the formazan crystals. The absorption was ultimately measured at 570 nm by using a Varioskan Flash multiplate reader (ThermoFisher, Massachusetts, USA), and the relative cell viability was calculated.
3
Cell Lysis and Enzyme Assays
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For the ALP, PK, and LDH activity assays, 1 ×106 cells were resuspended in 0.5 mL of ice-cold PBS and ultrasonically decomposed 5 times at 300 W for 5 s/time with 30s intervals on ice. For the HK activity assay, 5 ×106 cells were resuspended in one mL of extracting solution and ultrasonically decomposed. Then, the supernatant was collected at 8,000 g/min for 10 min at 4 °C. The supernatant was used for protein quantification by the BCA protein assay (KGP902; keyGEN, Nanjing, China) and subsequent activity assays according to the manufacturer’s instructions. The optical density values were determined at 450 wavelengths by a Varioskan Flash multi-plate reader (Thermo Scientific, Waltham, MA, USA). The mean values of the mean absorbance rates from three wells were calculated. Enzyme activities were calculated from optical density values and normalized by the total protein of each sample.
4
Quantifying HDAC Activity in Infected Macrophages
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The nuclear fractions of infected and uninfected macrophages were harvested as described earlier. They were used to perform the HDAC protein activity assay using Epiquik HDAC Activity Assay Kit (Fluorometric) (Epigentek, USA). The experiment was conducted as per the manufacturer’s instructions. Briefly, nuclear extracts (10 μg) were incubated with biotinylated HDAC substrate for 60 min at 37°C followed by incubation with capture antibody for 60 min and detection antibody for 30 min. Subsequently, the samples were incubated with Fluoro-Developer for 5 min at room temperature.
Furthermore, for the analysis of the activity of HDAC1, specifically in the reaction samples, 1 ug of immunoprecipitated HDAC1 protein obtained from infected and uninfected macrophages were used for the assay.
The amount of deacetylated histone, which is proportional to HDAC enzyme activity, was captured fluorometrically using Varioskan Flash Multiplate Reader (Thermo Fisher Scientific, USA) at 530 nm excitation and 590 nm emission. The enzymatic activities were calculated by plotting a standard curve using deacetylated standards provided with the kit. The unit of fluorometric absorbance used is RFU (Relative Fluorescence Unit) and was measured as;
Furthermore, for the analysis of the activity of HDAC1, specifically in the reaction samples, 1 ug of immunoprecipitated HDAC1 protein obtained from infected and uninfected macrophages were used for the assay.
The amount of deacetylated histone, which is proportional to HDAC enzyme activity, was captured fluorometrically using Varioskan Flash Multiplate Reader (Thermo Fisher Scientific, USA) at 530 nm excitation and 590 nm emission. The enzymatic activities were calculated by plotting a standard curve using deacetylated standards provided with the kit. The unit of fluorometric absorbance used is RFU (Relative Fluorescence Unit) and was measured as;
5
Improved CAS Assay for Cd-Chelating Ability
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A modified CAS liquid assay for the determination of Cd-chelating ability was improved from a Fe-CAS assay [66 (link)], which is conducted as follows: 4 mL 50 mM CAS, 2 mL 10 mM dipy, 1.5 mL 1 × 10−3 M HDPB, and 100 mM NaOH was titrated till the system turned green. After being placed stably for 5 min, a 5 mL buffer solution of sodium borate sodium hydroxide (pH 11.0) was added. Then diluted with H2O to a constant volume of 25 mL. The change of optical density value (OD) was measured at 602 nm within 40 min using a Varioskan Flash Multiplate Reader (Thermo Fisher, MA, USA). For the CAS plate assay, 2 g/100 mL agar was added to the solution system mentioned above. The chelation rate was calculated as (H2O-treated OD value–treatment OD)/(H2O-treated OD value).
Twenty AAs (1 mM) were respectively added to the CAS reaction system to determine the OD value. HLA-1-1 was cultured for 7 days with/without Thr to obtain the supernatant after centrifuging at 10,000 r/min for 10 min, then stored at −50 °C targeting concentrate supernatant via vacuum freeze-drying. Strains were harvested from fragments of HLA-1-1 obtained from the culture media after centrifuging at 10 000 r/min for 10 min, then put in the bead beater for milling a couple of times after adding magnetic beads.
Twenty AAs (1 mM) were respectively added to the CAS reaction system to determine the OD value. HLA-1-1 was cultured for 7 days with/without Thr to obtain the supernatant after centrifuging at 10,000 r/min for 10 min, then stored at −50 °C targeting concentrate supernatant via vacuum freeze-drying. Strains were harvested from fragments of HLA-1-1 obtained from the culture media after centrifuging at 10 000 r/min for 10 min, then put in the bead beater for milling a couple of times after adding magnetic beads.
6
Monitoring Bacterial Flagellar Secretion Dynamics
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The luciferase assay was performed as described before [18 (link)]. The plasmid pRG19 (motA::luxCDABE) was used for monitoring the switch from early substrate secretion to late substrate secretion. The class 3 gene motA is expressed only upon HBB completion. Synchronization of flagellar gene expression was achieved by placing the AnTc inducible tetA promotor upstream of the flhDC gene [44 (link)]. Cells were grown in white clear-bottom 96-well plates (Greiner) and production of light and the OD600 were measured over time using a Varioskan Flash multiplate reader (Thermo Fisher). After blank correction, relative light units (RLU) were calculated as:
7
Cell Viability Assay Optimization
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After different concentrations of drugs treatment, 20 μL MTS (Promega) was added to each well. The absorbance at 490 nm was recorded on a Varioskan Flash Multiplate Reader (Thermo Scientific) after incubation for 3 hours at 37°C. The ATP level of cell was determined by CellTiter-Glo Luminescent cell viability assay (Promega). Assays were performed in triplicate and repeated three times.
8
Cell Viability Assay Using CCK-8
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After exposure to EPs, the cell suspension was re-collected immediately, dispensed into 96-well plates, and incubated for 24 h. Cell viability was assessed using the cell counting kit (CCK)-8 commercial kit (Dojindo Molecular Technologies Inc., Kumamoto, Japan) following the manufacturer's instructions. The optical density (OD) value at a wavelength of 450 nm was measured using an automatic microplate reader (Varioskan Flash Multiplate Reader, Thermo Fisher Scientific, Waltham, MA, USA). Cell viability was calculated using the following equation:
where Aexp, Ablan, and Acon are the mean OD values of the experimental, blank medium, and control groups, respectively. For each parameter setting of each cell line, the experiment was repeated 10 times. All data were analyzed using GraphPad (9.0, GraphPad Software Inc., San Diego, CA, USA).
where Aexp, Ablan, and Acon are the mean OD values of the experimental, blank medium, and control groups, respectively. For each parameter setting of each cell line, the experiment was repeated 10 times. All data were analyzed using GraphPad (9.0, GraphPad Software Inc., San Diego, CA, USA).
9
Monitoring Protein-Lipid Interactions Using DMPC
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Interactions of the proteins with DMPC were monitored by the method of Zhu et al. (2005) [14 (link)], with slight modifications. Multilamellar liposomes (MLV) were prepared from a stock solution of DMPC in chloroform. DMPC was dried under an atmosphere of N2 and resuspended in 50 mM Tris-HCl buffer at pH 7.4, by vortexing for 3–5 min at 24 °C. The proteins (0.3 mg/mL) were incubated at 24 °C with DMPC MLV at a 3:1 (w/w) molar ratio (lipid:protein). The absorbance was monitored at 325 nm for 90 min using a Thermo Scientific Varioskan Flash multi plate reader at a temperature 24 °C.
10
Hemolysis Assay for Manganese-Curcumin Complexes
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The ability of
[MnII(Cur)2(HCur)] and HCur to induce hemolysis
was assessed according to a protocol described earlier.50 (link),74 (link) Briefly, fresh mice blood was collected and centrifuged at 1500
rpm for 10 min to remove blood plasma. The pellets of RBCs were washed
thrice in 35 mM PBS buffer and resuspended in 4% v/v of the same buffer.
In a 96-well plate, the compounds were serially diluted to a final
volume of 100 μL in each well. Next, 100 μL of RBC suspension
was added to each well and incubated at 37 °C for 1 h. The accompanying
DMSO used to dissolve [MnII(Cur)2(HCur)] was
also tested under identical conditions. After incubation, the plates
were centrifuged at 1500 rpm for 10 min. The supernatant (20 μL)
was added to 80 μL of PBS in a fresh 96-well plate. Hemoglobin
release was measured at 414 nm using a Thermo Scientific Varioskan
Flash Multiplate reader. For positive control, 0.1% Triton X-100 was
used, and for negative control, RBC in PBS buffer was used. Percentage
hemolysis was determined usingeq 11 .
The experiment was performed in accordance
with the guidelines of CPCSEA (Committee for the Purpose of Control
and Supervision of Experiments on Animals) and Institutional Animal
Ethics Committee (IAEC-23/2018) of Jawaharlal Nehru University, New
Delhi, India.
[MnII(Cur)2(HCur)] and HCur to induce hemolysis
was assessed according to a protocol described earlier.50 (link),74 (link) Briefly, fresh mice blood was collected and centrifuged at 1500
rpm for 10 min to remove blood plasma. The pellets of RBCs were washed
thrice in 35 mM PBS buffer and resuspended in 4% v/v of the same buffer.
In a 96-well plate, the compounds were serially diluted to a final
volume of 100 μL in each well. Next, 100 μL of RBC suspension
was added to each well and incubated at 37 °C for 1 h. The accompanying
DMSO used to dissolve [MnII(Cur)2(HCur)] was
also tested under identical conditions. After incubation, the plates
were centrifuged at 1500 rpm for 10 min. The supernatant (20 μL)
was added to 80 μL of PBS in a fresh 96-well plate. Hemoglobin
release was measured at 414 nm using a Thermo Scientific Varioskan
Flash Multiplate reader. For positive control, 0.1% Triton X-100 was
used, and for negative control, RBC in PBS buffer was used. Percentage
hemolysis was determined using
The experiment was performed in accordance
with the guidelines of CPCSEA (Committee for the Purpose of Control
and Supervision of Experiments on Animals) and Institutional Animal
Ethics Committee (IAEC-23/2018) of Jawaharlal Nehru University, New
Delhi, India.
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