Download Glycolysis Cell-Based Assay Kit

Glycolysis Cell-Based Assay Kit
Item No. 600450
Customer Service 800.364.9897 * Technical Support 888.526.5351
www.caymanchem.com
TABLE OF CONTENTS
GENERAL INFORMATION
3 Materials Supplied
4Precautions
4 If You Have Problems
4 Storage and Stability
4 Materials Needed but Not Supplied
INTRODUCTION
5Background
PRE-ASSAY PREPARATION
ASSAY PROTOCOL
Materials Supplied
Kit will arrive packaged as a -20°C kit. For best results, remove components and store as
stated below.
Item
Number
Item
Quantity/Size
Storage
6 About This Assay
600451
Glycolysis Assay Substrate
1 vial/250 µl
-20°C
7 Cell Culture Preparation
600452
Glycolysis Assay Enzyme Mixture
2 vials/lyophilized
-20°C
600453
Glycolysis Assay L-Lactate Standard
1 vial/120 µl
4°C
600454
Glycolysis Assay Cofactor
1 vial/250 µl
4°C
Cell-Based Assay Buffer Tablet
1 tablet
Room Temperature
8 Reagent Preparation
GENERAL INFORMATION
9 Standard Preparation
10 Plate Set Up
11 Performing the Assay
P ERFORMANCE CHARACTERISTICS 12Calculations
12 Performance Characteristics
RESOURCES
15Troubleshooting
16References
17 Related Products
10009322
If any of the items listed above are damaged or missing, please contact our Customer Service
department at (800) 364-9897 or (734) 971-3335. We cannot accept any returns without
prior authorization.
!
WARNING: This product is for laboratory research use only: not for
administration to humans. Not for human or veterinary diagnostic or
therapeutic use.
18 Warranty and Limitation of Remedy
19 Plate Template
20Notes
GENERAL INFORMATION
3
Precautions
Please read these instructions carefully before beginning this assay.
For research use only. Not for human or diagnostic use.
If You Have Problems
Technical Service Contact Information
Phone: 888-526-5351 (USA and Canada only) or 734-975-3888
Fax:734-971-3641
Email:[email protected]
Hours: M-F 8:00 AM to 5:30 PM EST
In order for our staff to assist you quickly and efficiently, please be ready to supply the lot
number of the kit (found on the outside of the box).
Storage and Stability
This kit will perform as specified if stored at -20°C and used before the expiration date
indicated on the outside of the box.
Materials Needed But Not Supplied
1. A plate reader with the capability to measure absorbance between 490-520 nm
2. A 96-well plate for culturing cells
3. A plate centrifuge
4. Adjustable pipettes and a repeating pipettor
5. A source of pure water; glass distilled water or HPLC-grade water is acceptable
4
GENERAL INFORMATION
INTRODUCTION
Background
Glycolysis is one of the major metabolic pathways in glucose metabolism. It is a process that
is conserved throughout evolution and occurs in virtually all cells. Glycolysis is the primary
source of ATP when oxygen is limiting, such as during intense strenuous exercise and also in
erythrocytes which lack mitochondria. The balance between gluconeogenesis and glycolysis
in responding to the needs of the entire body is tightly regulated by intracellular molecules
such as amino acids, and external factors such as insulin, which stimulates glycolysis. In
humans, various types of cells display different glycolytic pathways. For example, neurons
depend absolutely on aerobic glycolysis for energy whereas muscles derive almost all ATP
from anaerobic glycolysis.1 Over 80 years ago, Otto Warburg observed that cancer cells
preferentially obtained energy from aerobic glycolysis, which produces significantly higher
amounts of lactate from pyruvate, whereas normal cells use the aerobic tricarboxylic acid
cycle, which oxidizes pyruvate to carbon dioxide and water. In fact, the conversion of
glucose to lactate in the presence of oxygen is a critical aerobic pathway that allows cancer
cells to proliferate rapidly and the amount of lactate produced is correlated with tumor
aggressiveness.2,3 It is known that both embryonic stem cells and cancer cells share common
metabolic profiles with elevated glycolytic activity that might contribute to the proliferative
potential of these cells. Furthermore, the increase in glycolysis is partially mediated by
hypoxia-inducible transcriptional factor (HIF-1). However, the mechanism behind this
metabolic shift is not well understood.4
Lactate is the end product of glycolysis and is released into the extracellular environment.
Extracellular lactate levels are proportionally correlated with intracellular glycolytic activity
and thus have been used as metabolic biomarkers of tumors.2 In cancer research, direct
inhibiton of glycolysis has long been a chemotherapeutic strategy.5 Measuring lactate levels
is part of metabolomic approaches in drug discovery to monitor the effects of anticancer
drugs.
INTRODUCTION
5
About This Assay
Cayman’s Glycolysis Cell-Based Assay Kit provides a colorimetric-based method for
detecting L-lactate in culture medium. It can also be utilized to determine intracellular
lactate concentrations in cultured cells. In the assay, lactate dehydrogenase catalyzes the
oxidation of lactate to pyruvate, in which the formed NADH reduces a tetrazolium
substrate (INT) to a highly-colored formazan which absorbs strongly at 490-520 nm. The
amount of formazan produced is proportional to the amount of lactate released into the
culture medium and can be used as an indicator of cellular glycolytic rate. This single step,
no-wash assay can be adapted to high-throughput screening for glycolysis regulators at the
cellular level. Furthermore, the non-invasive sampling leaves the cells intact and thus allows
multiplex testing for additional markers.
6
INTRODUCTION
PRE-ASSAY PREPARATION
Cell Culture Preparation
1. Seed cells in a 96-well plate at a density of 104-105 cells/well in 120 µl of culture
medium and grow the cells overnight in a CO2 incubator at 37°C. If you are using
a culture medium containing serum, be sure to include a couple of wells containing
culture medium only (i.e., without any cells). These will be your background controls.
2.
The next day, treat the cells with or without compounds to be tested. We recommend
that each treatment be performed at least in duplicate.
3. Culture the cells in a CO2 incubator at 37°C for 24 hours, or for a period of time
according to your typical experimental protocol.
PRE-ASSAY PREPARATION
7
Reagent Preparation
Some of the kit components are in lyophilized or concentrated form and need to be
reconstituted or diluted prior to use. Follow the directions carefully to ensure proper
volumes of Assay Buffer are used to reconstitute or dilute the vial components.
1. Assay Buffer Preparation
Dissolve the Cell-Based Assay Buffer Tablet (Item No. 10009322) in 100 ml of
distilled water. This Buffer should be stable for approximately one year at room
temperature.
2. Glycolysis Assay Enzyme Mixture - (Item No. 600452)
The vial contains a lyophilized powder of enzymes. Immediately prior to use,
reconstitute the contents of the vial with 150 µl of Assay Buffer. If you are not using
all of the mixture at one time, make small aliquots and store at -80°C. Freezing and
thawing of this solution should be limited to a single time.
3. Reaction Solution
To make 12 ml of Reaction Solution sufficient for use on one 96-well plate, add
120 µl of each of the following mixtures to 11.64 ml of the Assay Buffer:
Glycolysis Assay Substrate (Item No. 600451)
ASSAY PROTOCOL
Standard Preparation
NOTE: Fetal bovine serum (FBS) contains LDH and thus will generate a high background
reading when used in the culture medium. We recommend that you use culture medium
containing a low percentage of FBS (such as 0.25-1%) or serum-free culture medium for your
experiments. When culture medium containing FBS is used, wells containing the same culture
medium without cells should be included in the assay as background controls.
Glycolysis Assay L-Lactate Standard
To prepare the standard for use in the glycolysis assay, obtain eight clean test tubes or
microcentrifuge tubes and label them #1 through #8. Aliquot 450 µl of Assay Buffer
into tube #1 and 250 µl into tubes #2-#8. Transfer 50 µl of the L-Lactic Acid Standard
(Item No. 600453) into tube #1 and mix thoroughly. The lactic acid concentration of
this standard, the first point on the standard curve, is 1 mM. Serially dilute the standard
by removing 250 µl for tube #1 and place into tube #2, mix thoroughly. Next, remove
250 µl from tube #2 and place into tube #3; mix thoroughly. Repeat this procedure for
tubes #4 through tubes #7. Do not add any standard to tube #8. This tube will be your
blank.
Glycolysis Assay Cofactor (Item No. 600454)
50 µl
250 µl
250 µl
250 µl
250 µl
250 µl
250 µl
Reconstituted Glycolysis Assay Enzyme Mixture (prepared in Step 2)
The Reaction Solution is stable for approximately one hour at room temperature.
S1
S2
250 µl
Assay
Buffer
450 µl
Assay
Buffer
10 mM
Bulk Standard
1
mM
S3
500
µM
S4
250 µl
Assay
Buffer
250
µM
S5
250 µl
Assay
Buffer
125
µM
S6
250 µl
Assay
Buffer
62.5
µM
S7
250 µl
Assay
Buffer
31.3
µM
S8
Final
Blank
250 µl
Assay
Buffer
250 µl
Assay
Buffer
15.7
µM
0
µM
Figure 1. Preparation of the L-Lactic Acid Standards
8
PRE-ASSAY PREPARATION
ASSAY PROTOCOL
9
Plate Set Up
Performing the Assay
There is no specific pattern for using the wells on the plate. However, a lactate standard
curve in duplicate has to be assayed with the samples. We suggest that each sample be
assayed at least in duplicate. A typical layout of standards and samples is given below (see
Figure 2). We suggest that you record the contents of each well on the template sheet
provided (see page 19).
5 6
8
9 10 11 12
S9
S9
S17 S17 S17 S25
•
Use different tips to pipette each reagent.
•
Before pipetting each reagent, equilibrate the pipette tip in that reagent
(i.e., slowly fill the tip and gently expel the contents, repeat several times).
•
Do not expose the pipette tip to the reagent(s) already in the well.
1
2
3
4
A
A
A
S1
S1
S1
B
B
B
S2
S2
S2 S10 S10 S10 S18 S18 S18 S25
C
C
C
S3
S3
S3
D
D
D
S4
S4
S4 S12 S12 S12 S20 S20 S20 S26
3. Add 90 µl of Assay Buffer to each well except standard wells.
E
E
E
S5
S5
S5 S13 S13 S13 S21 S21 S21 S26
4.
F
F
F
S6
S6
S6 S14 S14 S14 S22 S22 S22 S26
S9
7
Pipetting Hints
S11 S11 S11 S19 S19 S19 S25
G
G
G
S7
S7
S7
H
H
H
S8
S8
S8 S16 S16 S16 S24 S24 S24
S15 S15 S15 S23 S23 S23
A-H = Standards
S1-S26 = Sample Wells
1. Centrifuge the 96-well cell culture plate at 1,000 rpm for five miutes.
2. Using a new 96-well plate, transfer 100 µl of the standards prepared above into
appropriate wells. We recommend that the standards be run in duplicate.
Transfer 10 µl of supernatant from each well of the cultured cell plate to corresponding
wells on the new plate. (A smaller volume, 5 µl, of supernatant may be used if you
anticipate a high rate of glycolysis and you are using the L-Lactic Acid Standard to
calculate the level of glycolysis.)
5. Add 100 µl of Reaction Solution (prepared in Step 3, on page 8) to each well,
including the standard wells, using a repeating pipettor.
6. Incubate the plate with gentle shaking on an orbital shaker for 30 minutes at room
temperature.
7. Read the absorbance at 490 nm with a plate reader.
Figure 2. Sample plate format
10
ASSAY PROTOCOL
ASSAY PROTOCOL
11
The standard curve presented here is an example of the data typically produced with this
assay. However, your results will not be identical to these. You must run a new standard
curve with each experiment.
ANALYSIS
Calculations
0.5
1. Determine the average absorbance of each standard and sample.
2. Subtract the absorbance value of the blank from itself and all other standards and
samples. This is the corrected absorbance.
Plot the corrected absorbance values (from step 2 above) of each standard as a function
of the final concentration of lactic acid. See Figure 3, on page 13, for a typical standard
curve.
4.
Calculate the L-lactate concentration of the samples using the the corrected absorbance
of each sample and the equation below.
L-Lactate (µM) =
[
Absorbance - (y-intercept)
Slope
]
x 10*
*Multiply by 20 if using 5 µl of culture supernatant.
Performance Characteristics
Specificity:
To assess substrate specificity, the assay was performed with L-lactate replaced by structurally
similar compound D-lactate. No reaction occurred when D-lactate is used at a concentration
up to 10 mM.
12
ANALYSIS
Slope = 0.4243
y-intercept = 0.0027
r2 = 0.9987
0.4
Absorbance (490 nM)
3.
0.3
0.2
0.1
0.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
L-Lactate (mM)
Figure 3. L-Lactate Standard curve
ANALYSIS
13
RESOURCES
9
8
Troubleshooting
Lactic Acid Level (mM)
7
6
Problem
Possible Causes
Recommended Solutions
5
Erratic values; dispersion of duplicates
A. Poor pipetting/technique
B. Bubble in the well(s)
A. Be careful not to splash the
contents of the wells
B. Carefully tap the side of the
plate with your finger to
remove bubbles
No signal detected above background
in the sample wells
Sample was too dilute
Re-assay the sample using a
lower dilution
The absorbance values of the sample
wells were higher than the last
standard
Sample was too concentrated
Re-assay the sample using a
higher dilution
4
3
2
1
0
Control
Insulin
(1 µg/ml)
Figure 4. Insulin stimulates glycolysis in HEK293 cells.
HEK293 cells were seeded in a 96-well plate at a density of 50,000 cells/well in DMEM
containing 10% FBS and grown in a 5% CO2 cell culture incubator at 37°C. The next
day, cells were treated with vehicle or 1 µg/ml insulin in DMEM without FBS for
16 hours. Culture supernatant (10 µl) was removed from each well and the levels of
lactic acid were assayed according to the protocol described in the booklet. Insulin
at 1 µg/ml increased the production of extracellular lactic acid, indicating insulin
stimulates glycolysis in HEK293 cells.
14
ANALYSIS
RESOURCES
15
References
Related Products
1. Demetrius, L.A., Coy, J.F., and Tuszynski, J.A. Cancer proliferation and therapy: The
Warburg effect and quantum metabolism. Theoretical Biology and Medical Modeling
7, 2-14 (2010).
2. Griffin, J.L. and Shockcor, J.P. Metabolic profiles of cancer cells. Cancer 4, 551-561
(2004).
3. McCarty, M.F. and Whitaker, J. Manipulating tumor acidification as a cancer
treatment strategy. Altern. Med. Rev. 15(3), 264-272 (2010).
4. Kondoh, H., Lleonart, M.E., Nakashima, Y., et al. A common metabolic profile
shared between murine ES cells and primary cells bypassing senescence. Medical
Hypothesis and Research 4(1), 29-36 (2008).
5. Barger, J.F. and Plas, D.R. Balancing biosynthesis and bioenergetics: Metabolic
programs in oncogenesis. Endocrin-Related Cancer 17, R287-R304 (2010).
Adipogenesis Assay Kit - Item No. 10006908
Adipolysis Assay Kit - Item No. 10009381
Alanine Transaminase Activity Assay Kit - Item No. 700260
Annexin V FITC Assay Kit - Item No. 600300
Chloride Colorimetric Assay Kit - Item No. 700610
Coenzyme A Assay Kit - Item No. 700440
Glucose Colorimetric Assay Kit - Item No. 10009582
Glutathione Cell-Based Detection Kit (Blue Fluorescence) - Item No. 600360
Glycogen Assay Kit - Item No. 700480
JC-1 Mitochondrial Membrane Potential Assay Kit - Item No. 10009172
D-Lactate Assay Kit - Item No. 700520
L-Lactate Assay Kit - Item No. 700510
LDH Cytotoxicity Assay Kit - Item No. 10008882
Lipase Activity Assay Kit - Item No. 700640
Multi-Drug Resistance Assay Kit (Calcein AM) - Item No. 600370
Multi-Parameter Apoptosis Assay Kit - Item No. 600330
NAD+/NADH Cell-Based Assay Kit - Item No. 600480
Oxygen Consumption/Glycolysis Dual Assay Kit - Item No. 601060
Oxygen Consumption/MitoMembrane Potential Dual Assay Kit - Item No. 600880
Oxygen Consumption Rate Assay Kit (MitoXpress®-Xtra HS Method) - Item No. 600800
20S Proteasome Assay Kit - Item No. 10008041
Pyruvate Assay Kit - Item No. 700470
WST-1 Cell Proliferation Assay Kit - Item No. 10008883
WST-8 Cell Proliferation Assay Kit - Item No. 10010199
XTT Cell Proliferation Assay Kit - Item No. 10010200
16
RESOURCES
RESOURCES
17
18
RESOURCES
RESOURCES
H
G
F
E
A
For further details, please refer to our Warranty and Limitation of Remedy located on
our website and in our catalog.
D
Said refund or replacement is conditioned on Buyer giving written notice to Cayman
within thirty (30) days after arrival of the material at its destination. Failure of Buyer to
give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims
hereunder with respect to said material.
C
Buyer’s exclusive remedy and Cayman’s sole liability hereunder shall be limited to a
refund of the purchase price, or at Cayman’s option, the replacement, at no cost to Buyer,
of all material that does not meet our specifications.
B
Cayman Chemical Company makes no warranty or guarantee of any kind, whether
written or oral, expressed or implied, including without limitation, any warranty of
fitness for a particular purpose, suitability and merchantability, which extends beyond
the description of the chemicals hereof. Cayman warrants only to the original customer
that the material will meet our specifications at the time of delivery. Cayman will carry
out its delivery obligations with due care and skill. Thus, in no event will Cayman have
any obligation or liability, whether in tort (including negligence) or in contract, for any
direct, indirect, incidental or consequential damages, even if Cayman is informed about
their possible existence. This limitation of liability does not apply in the case of intentional
acts or negligence of Cayman, its directors or its employees.
1 2 3 4 5 6 7 8 9 10 11 12
Warranty and Limitation of Remedy
19
NOTES
This document is copyrighted. All rights are reserved. This document may not, in whole or
part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium
or machine-readable form without prior consent, in writing, from Cayman Chemical
Company.
©05/02/2014, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed
in U.S.A.
20
RESOURCES