Cells were plated on 24-well microplates and cultured for 24 h. Medium was collected and lactate determined by using colorimetric lactate oxidase-peroxidase assay lactate kit (Spinreact, Spain) as instructed by the manufacturer. The concentration of lactate in the medium was calculated by comparison with the signal obtained from standard with known amount of lactate provided by the manufacturer. Signals determined in medium without cells were also analyzed and subtracted from the amount determined in the presence of cells. ATP levels in cells were determined by using the CellTiter-Glo Luminiscent assay (Promega, USA), following the instructions of the manufacturer. Briefly, the same amount of reactive solution was added to culture medium to produce cell lysis with gentle movement. The mixture was homogenized and transferred to a white polystyrene 96-well assay plate. A standard with known amounts of ATP were also added in the same plate and mixed with reactive solution in a 1:1 ratio. A POLAR Star Omega fluorimeter and Omega Data Analysis Software (BMG Labtech) was used to analyze luminiscence. ATP amount was referred to the total of cells counted by hemocytometer after trypsin detachment seeded in a 24 well plate seeded in parallel.
Polarstar omega fluorimeter
Manufactured by BMG Labtech
The POLARstar Omega is a multi-mode microplate reader designed for fluorescence detection. It is capable of measuring fluorescence intensity, time-resolved fluorescence, and fluorescence polarization. The instrument features a monochromator-based optical system, allowing for flexible wavelength selection.
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3 protocols using polarstar omega fluorimeter
1
Lactate and ATP Quantification in Cultured Cells
2
Preparing Fibrillar Amyloid Proteins
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Lyophilized acetylated αS or βS was dissolved in 10 mM PBS (pH 7.4), and large aggregates were removed by centrifuge filtration (50 kDa MWCO, Millipore Sigma, St. Louis, MO). The dissolved protein was concentrated in 3 kDa centrifuge units (Millipore Sigma, St. Louis, MO) to 1 mg/mL (αS) or 3 mg/mL (βS). To create fibrils, 100 uL of each sample mixture was loaded into 96-well clear bottom plates (Corning, Corning, NY) with a single Teflon bead (3 mm, Saint-Gobain N.A., Malvern PA). The plates were sealed with Axygen sealing tape (Corning, Corning, NY) and shaken at 600 rpm and 37 °C in a POLARstar Omega fluorimeter (BMG Labtech, Cary, NC). Fibrils were allowed to form for at least 72 hours. Samples used for AFM, ESI-MS, ssNMR, and cell toxicity and shedding experiments were collected by centrifugation at 14k rpm for 2 hours, and washed through multiple rounds of re-suspension in 10 mM PBS (pH 7.4) and centrifugation at 14k rpm for 2 hours in order to remove residual soluble and non-fibrillar components.
3
Kinetics of Amyloid Aggregation of α-Synuclein
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Lyophilized native or glycated ac-αSyn was dissolved in PBS, passed through a 100 kD filter to remove large aggregates, and concentrated and washed using a 3 kD centrifugal filter. Thioflavin T (ThT) reactions consisted of 70 μM native or glycated ac-αSyn with 20 μM ThT in PBS. Where indicated, DJ-1 was added to samples at 140 μM. A total of 100 μL of the samples were aliquoted into clear-bottom 96-well plate with one Teflon bead to each reaction, sealed with Axygen sealing tape (Corning), and shaken at 600 rpm at 37 °C in a POLARstar Omega fluorimeter (BMG Labtech) for over 100 h. Fluorescence was monitored every 33 min.
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