Caspase-1 Detection Kit (Colorimetric) Manual BIOO RESEARCH PRODUCTS

BIOO RESEARCH PRODUCTS
Caspase-1 Detection Kit (Colorimetric) Manual
Catalog #: 5812
©BIOO Scientific Corp. • 2014
TABLE OF CONTENTS
GENERAL INFORMATION ........................................................................................ 1
Product Description ............................................................................... 1
Procedure Overview ............................................................................... 1
Kit Contents, Storage and Shelf Life ....................................................... 2
Required Materials/Equipment Not Provided With the Kit ..................... 2
Warnings and Precautions ..................................................................... 2
SAMPLE PREPRATION.............................................................................................. 3
Cell Culture Preparation ........................................................................ 3
CASPASE-1 ASSAY TEST PROTOCOL ..................................................................... 3
Assay Set-up/Reagent Preparation ......................................................... 3
Cell Lysate Preparation ......................................................................... 3
Assay Protocol ....................................................................................... 4
Data Analysis ......................................................................................... 4
TROUBLESHOOTING ................................................................................................ 5
Low Signal ............................................................................................. 5
High Signal ............................................................................................ 5
High Intra-Plate Variance (between Replicate Samples). ....................... 5
RELATED PRODUCTS ............................................................................................... 5
MaxDiscovery™ Caspase-1 Assay Kit is intended for laboratory use only, unless otherwise indicated. This product is
NOT for clinical diagnostic use. MaxDiscovery is a Trademark of Bioo Scientific Corporation (BIOO).
MaxDiscovery™ Caspase-1 Detection Kit (Colorimetric) - 5812
GENERAL INFORMATION
GENERAL INFORMATION
Product Description
Caspase-1 is an intracellular cysteine protease involved in inflammation. In resting cells, this
enzyme exists as an inactive zymogen known as procaspase-1 (molecular weight 45 kDa).
Intracellular procaspase-1 is activated when cells are externally stimulated by endotoxins and
other inflammatory stimuli. Induction of the inflammation eventually triggers activation of the
zymogen by proteolytic cleavage of the zymogen in the inflammasome to an active
heterodimer comprised of 20 kDa and 10 kDa subunits. The active caspase proteolytically
activates pro-IL-1β and pro-IL-18, thereby triggering the inflammatory action of these cytokines.
As such, caspase-1 is a validated therapeutic target for blocking inflammation. Therefore,
monitoring caspase-1 enzyme activity is a fundamental means to measure inflammation and in
the development of anti-inflammatory drugs.
The MaxDiscovery™ Caspase-1 Detection Kit (Colormetric) is a colorimetric microplate-based
assay to directly measure caspase-1 activation in cultured cells. This kit enables biomedical
researchers to determine caspase-1 levels in cultured cells from a broad range of cell lines.
The assay utilizes a simple, proven enzymatic assay to detect activated enzyme. The kit is
designed to be used with a microplate reader and contains sufficient materials to test 96
samples.
The unique features of the kit are:
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High sensitivity
High compatibility across a broad range of cell lines
A robust enzyme-based assay which does not require expensive instrumentation
High reproducibility
Procedure Overview
The MaxDiscovery™ Caspase-1 Assay Kit measures activated caspase-1 enzyme using a
direct, plate-based, colorimetric reaction. The kit contains a substrate solution containing a
caspase-specific tetrapeptide substrate, ac-Tyr-Val-Ala-Asp-pNA. The zymogen procaspase-1
has no reactivity towards this substrate. Active caspase-1 specifically cleaves the pNA group
from the peptide moiety. The free pNA group has a yellow color, which strongly absorbs light at
405 nm; the color is also visible in the wells.
caspase-1
ac-Tyr-Val-Ala-Asp-pNA + H20
Ac- Tyr-Val-Ala-Asp
+ pNA (yellow)
Mammalian cells are growth in culture wells and treated. The treated cells are lysed in the
wells (after removing the cell culture medium) using a cell lysis buffer provided with the kit. The
lysate is then directly added to wells in a 96 well reaction microplate (provided) containing the
ac-Asp-Glu-Val-Asp-pNA substrate solution. Caspase-1 activity is then measured using a plate
reader for the increase in absorbance at 405 nm in 30 minutes.
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MaxDiscovery™ Caspase-1 Detection Kit (Colorimetric) - 5812
Kit Contents, Storage and Shelf Life
The MaxDiscovery™ Caspase-1 Assay Kit has the capacity for 96 determinations or testing of
48 samples in duplicate. Store the kit at -20°C; the microtiter plate can be stored at -20°C, 4°C
or room temperature. When properly stored, the kit will maintain activity for 12 months after
receipt.
Kit Contents
Reaction Plate
Caspase-1 Substrate
Caspase-1 Reaction Buffer
Cell Lysis Buffer
1M DTT
Amount
96-well Plate (8 wells x 12 strips)
0.125 mL
10 mL
2 x 25 mL
0.1 mL
Storage
Room temp, 4°C, or - 20°C
- 20°C
- 20°C
- 20°C
- 20°C
Required Materials/Equipment Not Provided With the Kit
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Plate reader (405 nm)
Distilled or deionized water
Multichannel pipet (recommended)
Tissue culture supplies
Warnings and Precautions
BIOO strongly recommends that you read the following warnings and precautions to
ensure your full awareness of the techniques and other details you should pay close
attention to when running the assays. Periodically, optimizations and revisions are made to
the kit and manual. Therefore, it is important to follow the protocol included with the kit. If you
need further assistance, you may contact your local distributor or BIOO at
[email protected]
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Do not use the kit past the expiration date.
Try to maintain a laboratory temperature of (20–25°C/68–77°F). Avoid running assays
under or near air vents, as this may cause excessive cooling, heating and/or evaporation.
Also, do not run assays in direct sunlight, as this may cause excessive heat and
evaporation. Cold bench tops should be avoided by placing several layers of paper towel
or some other insulation material under the assay plates during incubation.
Use only distilled or deionized water since water quality is very important.
When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in
the lower corner of the well, making contact with the plastic.
BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its
products are made are of standard quality. There is no warranty of merchantability of this product, or of the
fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or
consequential damage, or expense arising directly or indirectly from the use of this product.
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MaxDiscovery™ Caspase-1 Detection Kit (Colorimetric) - 5812
SAMPLE PREPRATION
SAMPLE PREPARATION
The versatility of the MaxDiscovery™ Caspase-1 Kit (Colorimetric) allows users a great degree of
freedom in choosing their cell culture parameters.
Cell Culture Preparation
The suggested seeding concentration for most adherent cell lines is between 5.0 x 10 4 and 2.5
x 105 cells per mL. We suggest that users perform each experimental condition in either
duplicate or triplicate.
1.
2.
Seed cells in either a 96-well plate in 100 L of culture medium, or in a 24-well plate in
400 L of medium, or in a 6-well plate using 2 mL of medium.
Culture the cells in a CO2 incubator for at least 16 hours, and then replace the cell culture
media. Allow cells to grow to at or near confluence.
CASPASE-1 ASSAY
TEST PROTOCOL
CASPASE-1
ASSAY TEST PROTOCOL
Assay Set-up/Reagent Preparation
1. Turn on the plate reader, allow the light source to warm up, and set the absorbance
wavelength to 405 nm.
2. Warm up kit reagents to room temperature for 30 minutes.
3. Add 50 L of 1M DTT to 25 mL Cell Lysis Buffer and mix gently. Store on ice or at 4°C for
up to 6 hours and then discard any remaining buffer.
4. Dilute 100 L Caspase-1 Substrate (completely thawed) into 10 mL of Reaction Buffer.
Cell Lysate Preparation
Before you begin: This kit can be used with any adherent cultured mammalian cell line.
Culture the cells using typical cell culture conditions. After cell treatment, allow time for
stimulation to occur – some stimuli cause apoptosis in several hours whereas other stimuli
require several days to induce signal tranduction. Visually monitor cell morphology after
treatment to determine the best time point after treatment to observe caspase-1 activity.
1.
Gently remove the cell culture medium from the culture wells. Use caution at this step
especially if using nonadherent cells. Tilt the culture plates and aspirate the medium near
the side of the well (to minimize removing cells from the plate). Gently remove as much of
the medium as possible.
2.
Add the appropriate volume of Cell Lysis Buffer to each culture well:
Plate type
Lysis Buffer Volume (µL)
96-well
120
24-well
400
6-well
1000
3.
Gently shake, rock or tap the plate for 10 minutes to facilitate cell lysis and sample
homogenization.
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MaxDiscovery™ Caspase-1 Detection Kit (Colorimetric) - 5812
Assay Protocol
1. Add 100 L Caspase-1 Reaction Buffer to each well in the Reaction Plate.
2. Add 100 L of lysate to each well in the Reaction Plate. Mixing between the Caspase-1
Reaction Buffer and lysate is spontaneous.
3. Immediately measure the absorbance of each sample at 405 nm (Initial Reading). After
exactly 30 min, measure the absorbance again (Final Reading) .
Data Analysis
1. Calculate the absorbance change for each sample by subtracting the “initial reading” from
the “final reading”.
2. If samples were tested in duplicate, calculate the average absorbance change for each
condition.
3. The absorbance increase is proportional to the amount of active caspase-1 present in
each culture sample.
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MaxDiscovery™ Caspase-1 Detection Kit (Colorimetric) - 5812
TROUBLESHOOTING TROUBLESHOOTING
Low Signal
Possible Causes
Recommended Action
Not enough cells plated per well.
Stimulus not strong enough to
induce caspase-1 activation.
Lamp bulb expired.
Incorrect monochromator or filter
wavelength used.
Use a higher number of cells in each well.
Include a strong inducing control treatment in experimental design.
Replace bulb.
Use a wavelength at or near 405 nm.
High Signal
Possible Causes
Too many cells plated per well.
Reader was at wrong wavelength,
or reader was malfunctioning.
Recommended Action
1. Use a lower number of cells in each well.
2. Shorten the assay time interval from 30 minutes to 10 minutes.
Make sure the wavelength is 405 nm for the assay and read the plate again.
Verify reader calibration and lamp alignment.
High Intra-Plate Variance (between Replicate Samples).
Possible Causes
Inconsistent cell lysis.
Multichannel pipette was not
functioning properly.
Recommended Action
Allow Cell Lysis Buffer to warm up to room temperature prior to use and pipet
cells up and down several times to thoroughly lyse cells.
Verify pipette calibration and check that tips are on tight. Be sure all channels of
the pipette draw and dispense equal volumes.
RELATED PRODUCTS RELATED PRODUCTS
Apoptosis Assay Kits:
Product
Catalog Number
MaxDiscovery™ Caspase-3 Detection Kit (Colorimetric)
MaxDiscovery™ Caspase-3 Detection Kit (Flourimetric)
5811
5810
Cytotoxicity Assays:
Product
Catalog Number
MaxDiscovery™ Aspartate Transaminase (AST) Cytotoxicity Kit
MaxDiscovery™ Lactate Dehydrogenase Cytotoxicity Assay Kit
3460-11
3460-12
Protein Quantitation:
Product
Catalog Number
MaxDiscovery™ In Cell ELISA Kit
3440-02
Bioo Scientific Corporation
7050 Burleson Road
Austin, TX 78744 USA
Tel: 1.888.208.2246
Fax: (512) 707-8122
Made in USA
BIOO Research Products Group
[email protected]
[email protected]
www.biooscientific.com
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