TUNEL Two-Color Apoptosis Detection Kit Cat. No. L00429
(TRITC-labeled, for Flow
Cytometry)
Technical Manual No. TM0265 Version 03102011
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Description ………………………………………………………………. …………………
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Key Features
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Kit Contents
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IV
Storage..
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Protocol
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VI
Related Products
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VII
Troubleshooting
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VIII
Ordering Information…
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I. DESCRIPTION
TUNEL Two-Color Apoptosis Detection Kit (TRITC-labeled, for
Flow Cytometry
) (Cat. No. L00429) is used for
the fast detection of fragmented DNA in the nucleus during apoptosis. In this modified TUNEL assay kit,
fluorescein-labeled nucleotides binds with the DNA 3´-OH ends using natural or recombinant terminal
deoxynucleotidyl transferase (TdT or rTdT). The fluorescence could be detectd by Flow Cytometry.
II.
KEY FECTURES
Simplified Procedure: The kit contains ready-to-use reagents.
Enhanced Sensitivity: This kit can assay the cells during the early stages of apoptosis.
Enhanced Specificity: The kit can stain apoptotic cells.
Streamlined Process: The entire procedure takes about 3 hours.
High Veracity: The kit contains positive control reagent.
III. KIT CONTENTS
The TUNEL Apoptosis Detection Kit is available. L00429 is for detection using fluorescein Labeled nucleotides
(TRITC-5-dUTP).
Components
Cat. No.L00429
20 Assays
Cat. No.L00429
50 Assays
Cat. No. L00429
100 Assays
Storage Conditions
Equilibration Buffer
1 ml
2.5 ml
5.0 ml
-20°C
TRITC-5-dUTP
20 µl
50 µl
100 µl
-20°C
TdT
80 µl
200 µl
400 µl
-20°C
DAPI/RNase Buffer
10 ml
25 ml
50 ml
-20°C
2
DNase I (50 U/µl)
0.2 ml
0.5 ml
1 ml
-20°C
1X
DNase I buffer
0.2 ml
0.5 ml
1 ml
4°C
Note: Controls are not provided with kit
IV. STORAGE
Store the kit at -20°C. It will remain stable for one year.
V. PROTOCOL
Before use, order or prepare the following:
Fixation Solution: 1% paraformaldehyde in PBS, pH 7.4, freshly prepared.
Permeabilization Solution: 0.1% Triton X-100 and 0.1% sodium citrate in water, freshly prepared.
Note:
1. Please centrifuge the reagents in the kit before use.
2. Please prepare the proper amount of TUNEL Reaction Mixture according to the amount of the samples to save
reagent.
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Cell Samples
Rinse slides two times with PBS for five minutes each time.
Incubate with Blocking solution for 10 min at 15-25°C.
(
The Blocking solution contains 3% H
2
O
2
in methanol.
)
Rinse slides two times with PBS for five minutes each.
Fix cell samples with iced Fixation Solution for 30-60 mins at 4°C. (adjust the
concentration of cells to 1-2×10
6
cells/mL)
(The Fixation solution contains 1% Paraformaldehyde in PBS, pH 7.4, freshly prepared.)
Centrifuge and collect the cells at 300g for 5 min, rinse the cells with 5 mL of iced PBS for
twice
Discard the suspension (PBS), and resuspend the cell with residual PBS
Fix cell sample with 70% iced ethanol for more than 30 minutes or until before use at
-20°C. (adjust the concentration of cells to 1-2
×
10
6
cells/mL
)
Centrifuge and collect the cells at 300 g for 5 min, then rinse the cells with 1 mL of iced
PBS for twice.
Incubate in 1 mL Permeabilization Solution on ice (2-8°C) for two minutes.
(The Permeabilization Solution contains 0.1% Triton X-100 and 0.1% sodium citrate in
water, freshly prepared.)
Proceed as described in the Labeling Protocol.
Collect the cells
Centrifuge and collect the cells with 300g for 5 min, then rinse the cells with 1 mL iced PBS
for twice.
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Controls:
Negative control: Employ the cells or sections as described the labeling protocol. Label solution but do not add
any Terminal Deoxynucleotidyl Transferase (TdT) in TUNEL Reaction Mixture.
Positive control: Before beginning the labeling procedure, incubate the fixed and permeabilized cells or sections
with 100
μl
DNase I Solution for 10 -30 minutes at 21-37°C to induce DNA strand degradation.
DNase I Solution contains
10000 U/ml-20000 U/ml DNase I (grade I) depending on the sample to be stained in 1
×
DNase I buffer. One example of 1
×
DNase I buffer is 10 mM CaCl
2
, 6 mM MgCl
2
, and 10 mM NaCl in 40 mM
Tris-HCl, pH 7.9.
Labeling Protocol
The pretreated cells
Add 50 µl TUNEL Reaction Mixture to samples. Add a coverslip and incubate for 60
minutes at 37°C under wet conditions, protected from light.
(The TUNEL Reaction Mixture contains 45 µl Equilibration Buffer, 1 µl TRITC-5-dUTP
and 4 µl TdT, freshly prepared.)
Note: Add 50
μl
Label Solution to the negative control. To ensure a homogeneous dispersal of
TUNEL Reaction Mixture across the cell monolayer and to avoid loss to evaporation, the samples
should be covered with parafilm or a coverslip during incubation.
Rinse slides with PBS two times for five minutes each time.
Suspend the cells with 300-500ul DAPI/RNase Buffer at 15-25
℃
for 30 min, protected from light.
Analyse with Flow Cytometry: TRITC ( red fluorescence ) using excitation wave 543 nm and
emission wave 571 nm; DAPI ( blue fluorescence ) using ultraviolet excitation wave 340/380nm.
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VI. RELATED PRODUCTS
TUNEL Universal Apoptosis Detection Kit (Biotin labeled POD ), Cat. No. L00290
TUNEL Apoptosis Detection Kit for Adherent Cells (Biotin labeled POD ), Cat. No. L00296
TUNEL Apoptosis Detection Kit for Paraffin-embedded Tissue Sections (Biotin labeled POD), Cat. No. L00297
TUNEL Apoptosis Detection Kit for Adherent Cells (FITC labeled POD ), Cat. No. L00299
TUNEL Apoptosis Detection Kit for Paraffin-embedded Tissue Sections (FITC labeled POD), Cat. No. L00300
TUNEL Universal Apoptosis Detection Kit (For cell tissue section, TRITC-labeled), Cat. No. L00426
TUNEL Two-Color Apoptosis Detection Kit (FITC-labeled, for flow cytometry), Cat. No. L00428
TUNEL Universal Apoptosis Detection Kit (For cell tissue section, FITC-labeled), Cat. No. L00427
VII. TROUBLESHOOTING
TdT Dilution Buffer* contains 150 mM KCl, 1 mM 2-mercaptoethanol, and 50 % glycerol in 60 mM KPB, pH 7.2.
Problem
Step/Reagent
Possible cause
Solution
High
background
Fixation
Formalin fixation leads
to a yellowish stain in
cells containing melanin
precursors.
Use methanol for fixation. However, this
may lead to reduced sensitivity.
TUNEL reaction
The concentration of the
labeling mix is too high.
Reduce concentration of labeling mix from
10% to 50%.
Converter solution
There is endogenous
peroxidase activity.
Prior to cell permeabilization, block
endogenous peroxidase by incubating for 10
minutes in methanol containing 3% H
2
O
2
at
15-25°C.
Streptavidin-HRP has
engaged in non-specific
binding.
• Block with anti
-mouse serum.
• Block with PBS containing 3% BSA for 20
minutes.
• Reduce the concentration of
Streptavidin-HRP Solution to 50%.
The DAB incubation
time is too long.
Reduce the time of incubation.
Sample
Mycoplasma
contamination
Use a mycoplasma detection kit.
Highly proliferating
cells
Double staining with Annexin-V-Fluos* or a
similar substance.
Note: High background may make
measuring with microplate readers
impractical.
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Non-specific
staining
Fixation
After fixation, nuclease
activity is still high.
Block with the buffer containing dUTP and
dATP
TUNEL reaction
The concentration of
TdT is too high.
Reduce concentration of TdT from 10% to
50% with TdT dilution buffer*.
Low rate of
labeling
Fixation
Ethanol and methanol
can lead to diminished
labeling (chromatins are
not
cross-linked
with
proteins during fixation;
they are lost during the
procedure steps).
Fixate using 4% paraformaldehyde buffer,
formalin, or glutaraldehyde.
Extensive fixation leads
to excessive
cross-linkage with
proteins.
Reduce fixation time or fix by using 2%
paraformaldehyde PBS buffer (pH 7.4).
Permeabilization
The permeabilization
step is too short and the
reagents can’t reach
their target molecules.
• Increase the incubation time.
• Incubate at a higher temperature (such as
15-25°C).
• Optimize the concentration and action time
of proteinase K.
• Incubate with 0.1
M sodium citrate at 70°C
for 30 minutes.
No signal on
positive
control
DNase treatment
The concentration of
DNase I Buffer is too
low.
•
Incubate with 30000U/ml DNase I Solution*
or higher for 30 min at 37°C, and then rinse
with PBS.
Weak signals
Counterstaining
The dye is not suitable.
Counterstain with 5% methyl green in 0.1 M
veronal acetate, pH 4.0 or Hematoxilin.
VIII. ORDERING INFORMATION
TUNEL Two-Color Apoptosis Detection Kit (TRITC-labeled, for flow cytometry), Cat. No. L00429
GenScript
USA Inc
860 Centennial Ave., Piscataway, NJ 08854
Tel: 1-877-436-7274
Fax: 1-732-210-0262
E-mail:
product@genscript.com
Web: www.genscript.com
For Research Use Only.
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