The chaperone function of PfHsp70-xF, PfHsp70-xT and hHsp70 was investigated by monitoring their ability to suppress heat-induced aggregation of a model substrate, malate dehydrogenase (MDH) from the porcine heart (Sigma-Aldrich, USA) as previously described (Shonhai et al. 2008 (link); Zininga et al. 2016 (link)). Briefly, the capability of each protein to suppress thermally induced aggregation of MDH was monitored spectrophotometrically. MDH (0.6 μM) was added to the preheated buffer (50 mM Tris, pH 7.4, 100 mM NaCl) at 51 °C. The temperature was maintained at 51 °C for 80 min, and the absorbance was monitored at 340 nm in 5-min intervals using a SpectraMax M3 spectrometer (Molecular Devices, USA). BSA was used as a non-chaperone control in this assay. The data were analysed using GraphPad Prism 9.0 software (GraphPad Software, CA, USA).
Spectramax m3 spectrometer
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M3 is a multi-mode microplate reader capable of absorbance, fluorescence, and luminescence detection. It is designed for a wide range of applications in life science research and drug discovery.
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6 protocols using spectramax m3 spectrometer
1
Assessing chaperone function of Hsp70s
2
Neutralizing Antibody Inhibition Assay
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TZM-bl cells were maintained in TZM-bl medium as described above and cultured at 37 °C and 5% CO2. TZM-bl cells were plated in 96-well plates at a density of 1 × 105 cells per well in TZM-bl medium. The next day, the medium was removed and 100 μl of diluted serum from dolutegravir-treated or placebo-treated mice collected 7 day, 28 days, or 84 days post administration was added. Serum was diluted 1:20, 1:100, 1:500, 1:2500, 1:12,500, and 1:62,500 and incubated with cells for 30 min. The cells were infected with 100 μl of TZM-bl medium containing 40 µg/ml of DEAE-dextran and 3 × 103 TCIU of HIV-1JR-CSF per well was added. Approximately 48 h later, the medium was removed and the luciferase substrate One-Glo reagent (Promega, Madison, WI) supplemented with 0.2% Triton X-100 was added to allow for the measurement of luciferase activity and to inactivate virus. Luciferase was measured with a SpectraMax M3 Spectrometer (Molecular Devices, Sunnyvale, CA) and the results normalized to the luciferase activity of cells infected with HIV in the absence of serum and expressed as a percentage of decrease in luciferase activity. The TZM-bl cells were obtained through the NIH AIDS Reagent Program (catalog number 8129, passage number 2–6), cells were regularly checked for morphology by microscope.
3
Chaperone Activity of Hsp70 Proteins
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Hsp70 is capable of suppressing heat-induced aggregation of malate dehydrogenase (MDH) in vitro [3 (link)]. We thus investigated the chaperone activities of the GGMP mutant proteins versus that of wild type forms of PfHsp70-1 and DnaK by assessing their capability to suppress aggregation of heat-denatured malate dehydrogenase [MDH (Sigma-Aldrich, Darmstadt, Germany)] as previously described [41 (link),54 (link)]. First, it was important to validate the heat stability of the chaperones in the assay buffer and all the chaperone proteins were stable at 51 °C. However, under the same conditions, the substrate (MDH) aggregated spontaneously without chaperone at 51 °C and this reading was set as 100% aggregation. We mixed 0.2 μM of the chaperone. MDH was introduced into the reaction mix to achieve equimolar chaperone and MDH in 1:1 ratio as previously established [54 (link)]. Aggregation of MDH was monitored at 51 °C with readings taken every 5 min for 1 h. Absorbance was determined using a SpectraMax M3 spectrometer (Molecular Devices, San Jose, CA, USA). The assay was repeated in the presence of nucleotides (5 mM ATP/ADP).
4
Measuring HIV-1 Inhibition by Dolutegravir in TZM-bl Cells
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TZM-bl cells (NIH AIDS Research and Reference Reagent Program, catalog number 8129, passage number 2–6), were maintained at 37 °C 10% CO2 in TZM-bl medium. Cells were regularly checked for morphology by microscopy. TZM-bl cells were plated in 96-well plates (1 × 105 cells per well) in TZM-bl medium. The next day, the medium was removed and 100 μl of 1:100 dilutions of dolutegravir were added. Cells were incubated with DTG for 30 min and infected with 3 × 103 TCIU of HIV-1JR-CSF in 100 μl of TZM-bl medium containing 40 µg/ml DEAE-dextran. Approximately 48 h later, the medium was removed and the luciferase substrate One-Glo reagent (Promega, Madison, WI) supplemented with 0.2%Triton X-100 was added to allow for the measurement of luciferase activity and to inactivate virus. Luciferase activity was measured with a SpectraMax M3 Spectrometer (Molecular Devices, Sunnyvale, CA) and the results normalized to the luciferase activity of cells infected with HIV in the absence of DTG and expressed as a percentage of decrease in luciferase activity. Activity was measured in 3 independent experiments, each dilution measured in quadruplicate. The effective drug concentrations required to inhibit virus replication by 50% was calculated by fitting the data to a sigmoid dose-response curve with a variable slope using GraphPad Prism (GraphPad Software).
5
Fluorescence-based Cellular Assays
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All fluorescence and absorbance readings were taken on a Molecular Devices SpectraMax M3 Spectrometer. Confocal Laser Scanning Fluorescence Microscopy (CLSM) images were taken on a Zeiss LSM 510 Confocal Laser Scanning Microscope and processed using ImageJ software (with co-localisation analysis performed with the JaCoP plugin for ImageJ). All viability, ROS and GSH data analysis was performed using SigmaPlot v10.0 software.
6
Nanoparticle-Mediated Serotonin Assay
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Serotonin hydrochloride, Horseradish peroxidise (HRP), Monoamine Oxidase-A (MAO-A)FAD-bound (E.C:1.4.3.4) were purchased form Sigma-Aldrich. The Poly(amidoamine) nanoparticles (PAMAM) generations 4-7, were purchased from Sigma-Aldrich and manufactured by Dendritech Inc. The poly(propyl imine) (PPI) dendrimers generation 0-4, were purchased from SyMO-Chem. The Carboxy-H 2 DCFDA was purchased from Invitrogen. The 96 well plates used were purchased from TrueLine and all plates were analysed in a Molecular Devices SpectraMax M3 spectrometer.
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