Cells were seeded in a 60mm dish or 6 well plates. After the appropriate time point, cells were harvested, lysed in RIPA buffer (Thermo Instruments) and protein concentration was quantified by a lowry assay (using the Bio-Rad DC Protein Assay; #5000116 on a Spectramax device from Molecular Devices). Protein loading levels were equalized per lane and separated on SDS gels (Bio-Rad). Protein was transferred to a nitrocellulose membrance using semi-dry transfer (BioRad) Membranes were blocked with Omniblock (American Bio) and incubated with respected antibodies. When needed, secondary antibodies with HRP were incubated for 1hr. Enhanced chemiluminescent substrate for HRP enzymes was used to image protein levels (Thermo-Fischer; #34095) on a ChemiDoc MP imager with Soft Max Pro software (Biorad).
Spectramax device
Manufactured by Molecular Devices
The SpectraMax device is a multi-mode microplate reader designed for a variety of applications in life science research and drug discovery. It is capable of performing absorbance, fluorescence, and luminescence measurements on microplates.
Automatically generated - may contain errors
Lab products found in correlation
Semi dry transfer, by Bio-Rad (1 mentions)
Soft max pro software, by Bio-Rad (1 mentions)
Chemidoc mp imager, by Bio-Rad (1 mentions)
Verikine mouse ifn β elisa kit, by PBL Assay Science (1 mentions)
H2dcfda, by Molecular Devices (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Softmax software, by Molecular Devices (1 mentions)
4 protocols using spectramax device
1
Protein Quantification and Western Blot
2
Quantifying IFN-β in Cell Supernatant
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IFN-β released in cell supernatant was detected with Verikine Mouse IFN-β ELISA Kit (PBL, #42400-1) according to the manufacture’s protocol. Data were obtained from SpectraMax device (Molecular Devices).
3
ROS Quantification Using H2DCF-DA
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ROS generated were assessed using 2’,7’ - dichlorodihydrofluorescein diacetate
(H2DCF-DA; Sigma-Aldrich, St. Louis, MO, USA) staining, according
to the previous procedure [40 ].
H2DCF-DA stock solution was dissolved into anhydrous DMSO before
incubation, which was diluted to 1 mM and stored as aliquots in a -20 °C
freezer. The stock solution and aliquots were made in the dark. After the
treatments, the culture medium of groups (Figure
1 ) was collected and centrifuged at 9,9391 × g for 5
min. Fifty microliters of culture medium were separated and diluted three-fold
into PBS solution in a 96-well dark plate (SPL Life Science - Gyeonggi-do,
Korea). H2DCF-DA was added into each well at a final concentration of
25 µM and incubated for one hour at 37ºC. H2DCF-DA fluorescence
intensity was measured using a Spectramax device (Molecular Devices, CA, EUA).
The excitation filter was set at 480 nm and the emission filter at 530nm. The
results of each experiment were reported as mean values from triplicate wells as
arbitrary units.
(H2DCF-DA; Sigma-Aldrich, St. Louis, MO, USA) staining, according
to the previous procedure [40 ].
H2DCF-DA stock solution was dissolved into anhydrous DMSO before
incubation, which was diluted to 1 mM and stored as aliquots in a -20 °C
freezer. The stock solution and aliquots were made in the dark. After the
treatments, the culture medium of groups (
1
min. Fifty microliters of culture medium were separated and diluted three-fold
into PBS solution in a 96-well dark plate (SPL Life Science - Gyeonggi-do,
Korea). H2DCF-DA was added into each well at a final concentration of
25 µM and incubated for one hour at 37ºC. H2DCF-DA fluorescence
intensity was measured using a Spectramax device (Molecular Devices, CA, EUA).
The excitation filter was set at 480 nm and the emission filter at 530nm. The
results of each experiment were reported as mean values from triplicate wells as
arbitrary units.
4
Quantification of Amyloid-Beta Levels
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