(TG-5-RB) - Zen

Triglyceride Assay Kit-Bulk
500 Point Assay Kit, 96 well format
Cat# TG-5-RB
INSTRUCTION MANUAL
ZBM0014.05
STORAGE CONDITIONS

Reagents A & B, Buffers:
Store at 2 - 8C. Use reconstituted Glycerol Reagent A within 7 days.
Glycerol Standard
-20°C
ALL ZEN-BIO INC PRODUCTS ARE FOR RESEARCH USE ONLY. NOT APPROVED FOR HUMAN OR VETERINARY
USE OR FOR USE IN DIAGNOSTIC OR CLINICAL PROCEDURES.
LIMITED PRODUCT WARRANTY
This warranty limits our liability to replacement of this product. No other warranties of any kind,
expressed or implied, including without limitation, implied warranties of merchantability or fitness for a
particular purpose, are provided by Zen-Bio, Inc. Zen-Bio, Inc. shall have no liability for any direct, indirect,
consequential, or incidental damages arising out of the use, the results of use, or the inability to use this
product.
ORDERING INFORMATION AND TECHNICAL SERVICES
 Zen-Bio, Inc.
 3200 East NC-54 Suite 100
 PO Box 13888
 Research Triangle Park, NC 27709

Telephone
(919) 547-0692

Facsimile (FAX)
(919) 547-0693


Toll Free
Electronic mail (e-mail)
1-866-ADIPOSE (866)-234-7673
information@zen-bio.com

World Wide Web
http://www.zenbio.com
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The contents of this kit are sufficient for the assay of up to five 96 well plates (500 assay points). The
protocol is designed for assay of cells in a 96-well format. For other formats, please adjust the
volumes added to each well according to the surface area of the well/flask you are using. See your
cultureware manufacturer’s technical information for the specifications.
ITEMS INCLUDED IN THE KIT
Item
Wash Buffer
Lysis Buffer
Reagent A
Amount
250 ml
15 ml
40ml
11ml
QTY
1
1
1
1
Storage
4°C
4°C
4°C
10ml
2.5ml
1
1
4°C
4°C
100 l
3
-20°C
Standards
11 ml
Diluent
Other equipment/reagents required but not provided with the kit:
 Single-channel pipetter
 Multi-channel pipetter
 Plate reader with a filter of 540 nm
 Tubes to dilute glycerol standards
 Disposable reagent trays
 Mature adipocytes or other cells
 Clear bottom 96-well assay plates
1
4°C
Reagent B
Glycerol
standard
Rev 04.16.2014
Instructions
Reconstitute w/ 40 ml deionized water.
Reconstitute w/ 11 ml deionized water.
Combine and mix before use.
Use reconstituted Reagent within 7 days.
Reconstitute w/ 10 ml deionized water.
Reconstitute w/ 2.5ml deionized water
Combine and mix before use.
Glycerol @ 1mM [Reconstitute with 400 l
Standards Diluent to make the 200 M
glycerol standard; see manual for
recommended dilution scheme]
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ASSAY PROCEDURE
1. Warm the Wash buffer and Lysis buffer in a 37oC water bath.
2. Prepare the Reagent B by adding 10ml and
gently invert. DO NOT VORTEX! Use a
dissolved. Keep at room temperature. Store
Reagent B is stable for 60 days refrigerated
(2-8C).
2.5 ml deionized water to the labeled bottles and
pipet to ensure that the powder is completely
in a light protected bottle. Reconstituted Glycerol
(2-8C); store any remaining solution refrigerated
3. Remove all media from mature human adipocytes. Using about 15 ml of the wash buffer, wash
the cells one time with 150 µl wash buffer. Label the disposable tray “wash buffer” and retain
for later use.
4. Remove all Wash buffer. Using a disposable reagent tray (not provided), add 15 l Lysis buffer
per well. Incubate at 37oC - 50oC for 20 minutes.
5. After the incubation is complete, visually confirm cell lysis by checking the wells under a
microscope. If cells are not fully lysed, incubate for another 10 minutes. Sometimes pipet
mixing is necessary for full lysis.
6. Add 135 l warm Wash Buffer to each well.
7. Add 20 l Reagent B to each well. It is not necessary to mix at this time, however, gently tap
plate to help mix reagents. Incubate the plate at 37oC for 2 hours.
8. One hour prior to the assay, bring the glycerol standard and Reagent B to room temperature.
Warm the Standards Diluent to 37C. Prepare the standard curve as follows:
Pipette 400 l of the Standards Diluent into the 1 mM glycerol standard tube provided
and mix well by vortexing. This produces a diluted stock glycerol standard of 200 M.
Pipette 250 l of Diluent into 6 tubes (not provided). Using the newly diluted stock
glycerol solution, prepare a dilution series as depicted below. Mix each new dilution
thoroughly before proceeding to the next. The 200 M stock dilution serves as the
highest standard; the Diluent serves as the zero standard.
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400 l
Standards
Diluent
250 l
250 l 250 l 250 l 250 l
250 l
Std
200
M
100
M
50
M
25
M
12.5
M
6.25
M
3.125
M
Note: The above dilution series generates enough volume to perform the standard curve in duplicate. If
you wish to perform the standard curve in duplicate, please note that eight fewer data points can be
assayed with this kit.
9. Also at this time prepare the Reagent A by adding 40 ml and 11 ml respectively of deionized
water to the appropriate bottle and gently invert. DO NOT VORTEX! Use a pipet to ensure
that the powder is completely dissolved. Mix and combine the 2 volumes for use in the assay.
Keep at room temperature. If using a Glycerol Reagent A solution previously prepared and
stored at 2-8C, also bring to room temperature. Make sure there is enough Glycerol Reagent
A from one solution to treat all the points in the assay. It may be necessary to combine
solutions. Store in a light protected bottle. Reconstituted Glycerol Reagent A is stable for 7
days refrigerated (2-8C); store any remaining solution refrigerated (2-8C).
10. To a blank 96 well plate (not provided), add 80 l Wash Buffer to each well needed for the
assay (NOTE: do not add Wash Buffer to the wells used for the standard curve).
11. Working with one row or column at a time, mix the lysates very well using a multi-channel
pipet. Immediately transfer 20 l per well of the lysates to the corresponding well of the plate
containing the wash buffer. This results in a Dilution Factor of 5.
12. Prepare the standard curve. Pipet 100 l of each standard into a well. (NOTE: Eight wells are
necessary for the curve. If there are remaining wells on the assay plate, you can utilize the
remaining wells.)
13. Add 100 l Reagent A to samples and standards. Mix by pipetting up and down one time.
Incubate at room temperature for 15 minutes.
14. Read at 540 nm using a microtiter plate reader.
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GLYCEROL STANDARD CURVE
This kit is designed to show relative lipid accumulation of experimental treatments compared to
controls. The assay is based on the equation
1 M Triglyceride yields 1M glycerol + Free Fatty Acids
The reagent measures the concentration of glycerol released after lysing the cells and hydrolyzing the
triglyceride molecules. The triglyceride concentration can then be determined from the glycerol
values.
Generate standard curve: see example below
[DO NOT use this standard curve to generate your data. This is an example.]

Subtract the OD value of the 0M standard from all OD values including the standard curve.
Glycerol
(uM)
0
3.125
6.25
12.5
25
50
100
200
OD
0.048
0.059
0.07
0.098
0.127
0.2
0.353
0.649
slope
intercept
2
R
0.003
0.053
0.9997
OD
0.048
0.058
0.07
0.098
0.13
0.205
0.362
0.667
ODblank
ODblank
0.011
0.022
0.05
0.079
0.152
0.305
0.601
0.01
0.022
0.05
0.082
0.157
0.314
0.619
Avg
ODblank
0.048
0.059
0.070
0.098
0.129
0.203
0.358
0.658
y = observed O.D. minus the blank
x = concentration of glycerol in M
To calculate x for each y, (i.e. to change the observed O.D. into glycerol concentration) use the
following equation:
y=(slope) times (x) plus intercept
y=mx+b so x=(y-b)/m
x=(y – 0.0006)/0.0014 where 0.0014= slope of the line and 0.0006= y intercept.
Be careful to enter the proper sign for the y intercept value as it may be a negative number.
Any OD values greater than the highest standard (200 M) should be suspect. The compound should
be re-assayed using a lower dose of the compound at treatment OR a dilute solution of the condition
medium at the time of the assay.
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The R2 value should be equal or greater then 0.98 for the standard curve to be valid. Any R2
values below 0.98, must have the standard curve run again.
Solve for the Total Glycerol concentration (i.e. total triglyceride concentration) for each OD.
Remember to include the Dilution Factor in the equation.
Data is expressed as M Glycerol.
NOTE: Any OD values that are negative after the blank is subtracted should be considered to be 0
for the OD value.
FREQUENTLY ASKED QUESTIONS
1. Can I buy the reagents separately? The only reagents sold separately are Glycerol
Reagent A (cat# RGTA-10; RGTA-40) and the glycerol standard for the Triglyceride
Assay kit (cat# TG-GLYSTAN).
2. Can I use another plate format besides 96 well? This kit is designed for the assay of
A 96 well plate (100 assay points). We do not have a protocol for other formats.
3. Can I use this kit to measure total triglyceride in other cell lines and other human
and non-human cells? Yes. The assay is not species specific. As long as the sample
concentration is in the linear range, this kit should be able to detect it.
4. My cells did not lyse. What can I do? If cells are not fully lysed, incubate for another
10 minutes at 37oC - 50oC. Sometimes mixing by pipetting up and down several times
is necessary for full lysis.
5. I do not have time to complete the assay. Can I freeze the samples? Yes. The cell
lysates can be stored at -80C for a maximum of 7 days. Mix the thawed lysates in the
plate by pipetting up and down several times. Allow all reagents and samples to reach
room temperature BEFORE adding the Wash Buffer and Glycerol Reagent A to
complete the assay.
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TROUBLESHOOTING
Problem
High background or the triglyceride
reagent turns a darker color before
the assay begins.
Suggestions


Use clean tray and tips
Change pipet tips frequently
Edge effects

Ensure a saturated humidity in the incubator
to prevent evaporation from the outside wells
Inconsistent OD reading

Be careful when pipetting to avoid bubbles. If
bubbles persist, burst the bubbles using a
large gauge needle prior to reading and read
the plate again.

Mix the lysates well before transferring the
20l to the Wash buffer plate.
REFERENCES
1. Green, H. and Kehinde, O. (1974) Sublines of mouse 3T3 cells that accumulate lipid. Cell 1,
113-116.
2. Hauner, H., et al., (1989) J. Clin. Invest.(84), 1663-1670.
3. Kuri-Harcuch W, Wise LS, Green H. (1978) Interruption of the adipose conversion of 3T3 cells
by biotin deficiency: differentiation without triglyceride accumulation. Cell 14:53-58.
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APPENDIX A: PLATE LAYOUT
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9
10
11
12
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APPENDIX B: TRIGLYCERIDE ASSAY FLOWCHART
All media
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
Remove all media from wells.
150 l Wash Buffer
150 l Wash Buffer
Wash with 150 l Wash Buffer
Remove all Wash Buffer from wells
and add 15 l Lysis Buffer/well.
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
Add 15 l Lysis
Buffer
o
Incubate 20 minutes at 37 – 50 C
Verify lysis and add 135 l warm
Wash Buffer. Mix by pipetting up and
down 3 times.
Add 135 l warm
Wash Buffer
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
Add 20 l Reagent B
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
Add 20 l Reagent B and incubate 2
o
hours at 37 C.
One hour prior to assay,
reconstitute Glycerol Reagent A and
prepare standards. Keep all at room
temperature.
Add 80 l Wash
Buffer
Add 80 l Wash Buffer to a new plate.
Mix lysates and transfer 20 l lysates
to the wells containing Wash Buffer.
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
20 l
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
O
O
O
O
O
Transfer 100 l of each standard to a
new plate.
Standards
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
Add 100 l Glycerol Reagent A to
samples and standards.
GLYCEROL
REAGENT A
OOO
OOO
OOO
OOO
OOO
An additional blank assay plate
may be necessary for the assay of
glycerol standards.
o
Incubate 15 minutes at 25 C (room
temperature). Pop the bubbles in
each well.
Measure the optical density of each
well at 540 nm using a
spectrophotometer plate reader.
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