Product name :QuantiFluoÔäó Caspase-3 Assay Kit

Catalog Number :DCS3-100

Quantity :100 tests

Price :173 Eur

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Supplier :Bioassays

DESCRIPTION
Caspases are members of the aspartate-specific cysteinyl protease family
that play a central role in apoptosis. Apoptosis is involved in a variety of
physiological and pathological events, ranging from normal fetal
development to diseases such as cancer, organ failure, and
neurodegenerative diseases. Caspase-3 is key biomarker in the
assessment of apoptosis and in understanding mechanism of apoptosis
induction.
BioAssay Systems’ Caspase-3 Assay Kit provides a convenient means to
measure caspase-3 activity in biological samples. In the assay, a specific
substrate (N-Ac-DEVD-AFC) is cleaved by active caspase-3, forming a
highly fluorescent product. The fluorescence intensity (lexc/em = 400/490nm)
is proportional to the caspase-3 activity.
KEY FEATURES
Safe. Non-radioactive assay.
Convenient and high-throughput. Homogeneous "mix-incubate-measure"
type assay. Can be readily automated on HTS liquid handling systems for
processing thousands of samples per day.
APPLICATIONS
Determination of caspase-3 activity in cell and tissue lysate.
HTS screening for apoptosis inducers and inhibitors.
KIT CONTENTS
Assay Buffer: 12 mL Substrate: 240 μL
DTT Solution: 240 μL
Storage conditions: This product is shipped at room temperature. Upon
delivery, store all reagents at -20°C. Shelf life of 12 months after receipt.
Precautions: reagents are for research use only. Normal precautions for
laboratory reagents should be exercised while using the reagents. Please
refer to Material Safety Data Sheet for detailed information.
96-WELL ASSAY PROCEDURE
Note: The following procedure is for standard assays in a 96-well plate. If
cells are cultured in plates other than a 96-well plate, it is necessary to
prepare the cell lysate (See General Considerations). Duplicate assays are
recommended for controls and samples.
1. Cell culture. Seed 100 μL of 1,000 to 100,000 cells into wells of a sterile
black clear-bottom 96-well plate (Note: The cell number to be used
depends on the cell line). Incubate overnight at 37°C in a cell culture
incubator.
2. Cell treatment. Add 10 μL test compounds (e.g. apoptosis inducers or
inhibitors) at desired concentration, and 10 μL vehicle (medium in which
the test compound is dissolved) as a control.
Incubate cells for a desired period of time.
3. Caspase-3 Assay. Prior to assay, bring all reagents to room temperature.
Briefly centrifuge tubes. Prepare enough Working Reagent for all assay
wells. For each well, mix 100 μL Assay Buffer, 2 μL Substrate and 2 μL
DTT.
Remove culture media from assay wells. For adherent cells, simply
aspirate the culture media from wells. For suspension cells, centrifuge
cells for 5 min at 500 x g, carefully remove media by aspiration.
Immediately add 100 μL Working Reagent to each assay well. Mix the
reagents completely by shaking the plate for 60 sec at 100-200 rpm on a
plate shaker. The Working Reagent lyses cells and supports optimal
caspase-3 activity.
Incubate plate at 37°C for 60 min in the dark.
4. Read fluorescence intensity at lexc/em = 400/490 nm.
Note: If the caspase-3 activity is low, increase the incubation time, or use
more cells.
CALCULATION
Subtract the fluorescence intensity values from that of the control wells. The
DF values represent the relative caspase-3 activity.
GENERAL CONSIDERATIONS
For cells not cultured in a 96-well plate, cell lysates are prepared separately
and used for the caspase assay.
After cells have been treated with test compounds for the desired period of
treatment, remove culture medium. For adherent cells simply aspirate the
culture medium. For suspension cells centrifuge cells at 500 x g for 5 min
and aspirate the medium.
Lyse cells by adding 300 μL per 106 cells of 50 mM Hepes (pH 7.2), 100
mM NaCl, 0.5 % (v/v) Triton X-100.
Shake the cell suspension on for 30 min at 4°C.
Centrifuge the cell suspension at 2,500 x g for 10 min at 4°C.
Transfer supernatants to clean tubes. If not assayed on the same day,
lysates can be stored at -80°C for one month.
To assay, add 50 μL of sample lysates into separate wells of a black 96-well
plate. Immediately add 100 μL Working Reagent to each assay well.
Incubate plate at 37C for 60 min in the dark. Read fluorescence intensity
lexc/em = 400/490 nm.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipetting devices, centrifuge tubes, black flat bottom 96-well plates and
plate reader.

datasheet of product :QuantiFluoÔäó Caspase-3 Assay Kit QuantiFluoÔäó Caspase-3 Assay Kit

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