TUNEL細胞凋亡檢測試劑盒

品牌：上海銘博 | 貨號：T2190

Cat.: T2190TUNEL Apoptosis Assay Kit

Size:50T

Kit Components:

Components                                                   Amount

Component A: 100X TF3-dUTP                  1 vial (25 uL)

Component B: Reaction Buffer                  1 bottle (5 mL)

Component C: 1000X Hoechst                   1 vial (50 uL) 

Introduction:

DNA fragmentation represents a characteristic of late stage apoptosis. DNA fragmentation in apoptotic

cells can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling

(TUNEL). The TUNEL assay relies on the presence of nicks in the DNA which can be identified by TdT,

an enzyme that catalyzes the addition of dUTPs that are secondarily labeled with a marker. All the existing

TUNEL assays contain the highly toxic sodium cacodylate which might induces apoptosis and also

decrease DNA production and DNA strands. Our TUNEL Apoptosis Assay Kit uses proprietary buffer

system free of sodium cacodylate. The kit is based on incorporation of a fluorescence dye TF3 modified

deoxyuridine 5 -triphosphates (TF3-dUTP) at the 3 OH ends of the DNA fragments that form during

apoptosis. The assay is optimized for the direct detection of apoptosis in either detached or attached cells

without using antibody.

The kit provides all the essential components with an optimized assay protocol. It is suitable for

fluorescence microplate reader, fluorescence microscope, or flow cytometer. Its signal can be easily

detected at Ex/Em = 550nm/590 nm.

Assay Protocol:

1. Culture cells to an optimal density for apoptosis induction according to your specific protocol. We

recommend about 30,000 to 50,000 cells/well for adherent cells grown in a 96-well microplate culture, or

about 1 to 2 x 106 cells/mL for non-adherent cells. At the same time, culture a non-induced negative

control cell population at the same density as the induced population for every labeling condition. Here are

a few examples for inducing apoptosis in suspension culture:

1) Treat Jurkat cells with 2 g/ml camptothecin for 3 hours.

2) Treat Jurkat cells with 1 M staurosporine for 3 hours.

3) Treat HL-60 cells with 4 g/ml camptothecin for 4 hours.

4) Treat HL-60 cells with 1 M staurosporine for 4 hours.

2. Fixation and Permeabilization

2.1 Remove cell media. 

2.2 Add 100 L/well/96-well plate of 4% formaldehyde fixative buffer (not supplied) to each well.

Note: For non-adherent cells, add desired amount (such as 2 x 106 cells/mL) of 4% formaldehyde fixative buffer. 
 2.3 Incubate plates for 20 to 30 minutes at room temperature.

2.4 Remove fixative.

Optional: add 100 L/well/96-well plate of the permeabilization reagent (0.2% Triton X-100 in PBS, not

supplied) after the fixation if needed, and incubate the plate for 10 minutes at room temperature.

2.5 Wash the cells with PBS 2-3 times.

Optional: You may also prepare a positive control for TUNEL reaction using DNAase I by digesting cells

with DNAase I for 30 min at room temperature before proceed to TUNEL reaction (Step 3)

3. TUNEL reaction

3.1 Prepare reaction mixture just before use based on the number of samples to be assayed:

Reaction Components                          Volume Per Well

100X TF3-dUTP (Component A)                  0.5 uL

Reaction Buffer (Component B)                    50 uL

Total volume                                                50.5 uL 

Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density.

3.2 Add 50 uL of the reaction mixture (from Step 3.1) to each well or tube and incubate at 37℃ for 60

minutes. 

 3.3 Remove the reaction mixture, and wash the cells 3-5 times with 200 uL/well of PBS.

4. Monitor the fluorescence intensity by fluorescence microscope, flow cytometer, or fluorescence

microplate reader at Ex/Em = 550/590 nm.

5. Optional: Stain the nucleus with 1X Hoechst (Component C, Ex/Em = 350/460 nm) for image analysis.

Data Analysis:

1. 96-Well Fluorescence Plate Reader Sample data:
 

Figure 1. Apoptosis analysis in Hela cells using TUNEL Apoptosis Assay Kit. Hela cells at 30,000

cells/100 L/well were treated with 1 M staurosporine for 4 h (Red) while un-induced cells were used as a

control (Blue). Cells were incubated with reaction mixture for 1 hour at 37℃. The Fluorescence was

measured at Ex/Em = 550/590 nm (cut off at 570 nm) with a Flex Station microplate reader using bottom

read mode. 
2. Fluorescence Microscope Sample data: 
                      A. Control                                                                          B. 2h                                                                      C. 4h

Figure 2. The Fluorescence imagining indicated the increase in TUNEL reaction with the addition of 1 M

staurosporin for 2h (B) or 4h (C) compare to control (A) in Hela cells. Cells were incubated with reaction

mixture for 1 hour at 37℃. The Fluorescence intensity of the cells (30,000 cells/ 100 L per well) was

analyzed under a fluorescence microscope with a TRITC channel. DNA strand breaks are shown as more

intense fluorescent staining spots in cells treated with staurosporin.

References

1. Gavrieli.Y, Sherman Y., Ben-Sasson S.A. 1992. Identification of programmed cell death in situ via

specific labeling of nuclear DNA fragmentation.J Cell Biol. 119(3):493-501.

2. Huerta S., Goulet E.J., Huerta-Yepez S., Livingston E.H.,2007. Screening and detection of apoptosis.

J Surg Res. 139(1):143-56.

3. Webster K., ParishJ, Pandya M., Stern P.L., Clarke A.R., and Gaston K. 2000. The human

papillomavirus (HPV) 16 E2 protein induces apoptosis in the absence of other HPV proteins and via a

p53-dependent Pathway* J Biol Chem. 275(1): 87-94.

4. Whiteside G., Cougnon N., Hunt S.P., and Munglani R. 1998. An improved method for detection of

apoptosis in tissue sections and cell culture, using the TUNEL technique combined with Hoechst stain.
Brain Res Brain Res Protoc 2: 160 164. 

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