ody was added to the plate, with the final concentration
per well ranging from 400 ng/mL down to 0 ng/mL of antibody. The plates
were then incubated at 37C / 5% CO2
for a total of 5 hours
to induce apoptosis. 3 hours into the 5 hour treatment, CellTiter-Blue
reagent was added to each well with the Equator (note: for plates later
3/7 reagent, CellTiter-Blue
1:4 in 1X PBS before addition to the assay plate). After the CellTiter-Blue
addition, plates were incubated for 2 hours at 37C / 5% CO2
When the 5 hour incubation with anti-FAS antibody was complete, fluorescence
was recorded at excitation 540 nm and emission of 590 nm with the PHERAstar.
The caspase reagents were then added to the plates with the Equator. Apo-ONE
was added to one plate containing CellTiter-Blue
reagent, and the
plate was incubated at room temperature for 1 hour, followed by fluorescence
reading with the PHERAstar at excitation 485 nm and emission of 520 nm.
reagent was added to the plate receiving the diluted
reagent, and incubated for 1 hour at room temperature.
Luminescence was then recorded with the PHERAstar.
For the 1536-well assay format, a density of 4,000 cells per well was
plated with the Deerac Fluidics Equator. The remaining multiplex protocols
were performed identically to the low-volume 384 protocols listed above.
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Results and Discussion
Promegas CellTiter-Blue assay multiplexed with either Caspase-Glo
3/7 (figure 2) or Apo-ONE assay was prepared in both low-volume 384-
and 1536-well format (f
. Promegas Multiplexed Luciferase Reporter and Cell Viability Assays performed on the PHERAstar2
. Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma3
. Cell Proliferation and Viability Measurement4
. LIVE/DEAD Viability/Cytotoxicity Assay for Animal Cells Using the SpectraMax Gemini XS Fluorescence Microplate Reader (MaxLine Application Note #43)5
. RT-PCR Primers for the Study of Apoptosis6
. A Further Step in Understanding Apoptosis
Direct Detection of PARP Cleavage7
. Apoptosis detection by annexin V and
active caspase-3 with the Agilent 2100
. Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays9
. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations10
. Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success11
. Using Validated siRNAs in Functional Genomic Assays