Tunel protocol For Cells On Coverslips



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TUNEL Protocol For Cells On Coverslips

The following protocol was developed using HeLa cells treated with 0.5 µM staurosporine for 4 hours to induce apoptosis. Cell type and treatment may influence the number of apoptotic cells detected. This protocol uses the Click-iT® TUNEL Alexa Fluor® Imaging Assay Protocol from Life Technologies.



1. Cell Fixation and Permeabilization

This protocol is optimized with a fixation step using 4% paraformaldehyde in PBS followed by a permeabilization step with 0.25% Triton®X-100, but is amenable to other fixation and permeabilization reagents such as 70% ethanol.

  1. Remove media and wash coverslips once with PBS. Note: If there is a chance that cells maybe lost by this wash step, proceed directly to fixation (step 2) without performing the wash step.

  2. Add a sufficient volume of fixative (4% paraformaldehyde) to completely cover the coverslips.

  3. Incubate samples for 15 minutes at room temperature.

  4. Remove fixative.

  5. Add sufficient volume of the permeabilization reagent (0.25% Triton® X-100 in PBS) to completely cover the coverslips.

  6. Incubate samples for 20 minutes at room temperature. Then wash twice with deionized water.

2. Preparing a Positive Control (Optional)

The DNase I generates strand breaks in the DNA to provide a positive TUNEL reaction. There is sufficient DNase I to perform ~18 positive controls.

  1. Wash coverslips with deionized or molecular biology grade water.

  2. Prepare DNase I solution according to Table 3 and mix well. Note: Do not vortex the DNase I solution as DNase I denatures with vigorous mixing.




Table 3. DNase I Solution.

Reaction Components

Number of Coverslips




1

2

3

Deionized water

89 µL

178 µL

267 µL

DNase I buffer (Component H)

10 µL

20 µL

30 µL

DNase I (Component G)

1 µL

2 µL

3 µL

Total Volume

100 µL

200 µL

300 µL



  1. Add 100 µL of the DNase I solution (prepared in step 2) to each coverslip and incubate for 30 minutes at room temperature.

  2. Wash coverslips once with deionized water, proceed to TdT Reaction.


3. TdT Reaction

This protocol uses 100 µL of the TdT reaction cocktail per coverslip. It is important toequilibrate cells to maximize efficiency of the TdT reaction.

  1. Add 100 µL TdT reaction buffer (Component A) to each coverslip and allow the solution to spread completely over the surface. 

  2. Incubate coverslips for 10 minutes at room temperature.

  3. Remove TdT reaction buffer.

  4. Prepare the TdT reaction cocktail according to Table 4.


Table 4. TdT Reaction Cocktails.

Reaction Components

Number of Coverslips




1

2

4

5

10

TdT reaction buffer (Component A)

94 µL

188 µL

376 µL

470 µL

940 µL

EdUTP (Component B)

2 µL

4 µL

8 µL

10 µL

20 µL

TdT* (Component C)

 4 µL

8 µL

16 µL

20 µL

 40 µL

Total Volume

100 µL

200 µL

400 µL

500 µL

1 mL

*Immediately after using the TdT enzyme (Component C), return the vial =–20°C. Do not keep the enzyme at room temperature.



  1. Add 100 µL of the TdT reaction cocktail (prepared in step 4) to each coverslip and allow the solution to spread completely over the surface.

  2. Incubate coverslips for 60 minutes at 37°C. Use a humidified chamber to protect against evaporation. Remove the reaction cocktail.  Note: This reaction can be performed overnight at room temperature.

  3. Wash coverslips twice with 3% BSA in PBS for 2 minutes each.

4. Click-iT® Reaction

  1. Prepare the Click-iT® reaction buffer additive (Component E) by adding 625 µL of deionized water to the Component E vial. After use, aliquot and store any remaining solution at =–20°C.  When stored as directed the stock solution is stable for up to 1 year.

  2. Prepare the Click-iT® reaction cocktail according to Table 5 and mix well by vortexing. Note: Use the Click-iT® reaction cocktail within 15 minutes of preparation.

  3. Immediately add 100 µL of the Click-iT® reaction cocktail (prepared in step 2) to each coverslip and allow the solution to spread completely over the surface.


Table 5. Click-iT® Reaction Cocktails.

Reaction Components

Number of Coverslips




1

2

4

5

10

Click-iT® reaction buffer (Component D)

97.5 µL

195 µL

 390 µL

487.5 µL

975 µL

Click-iT® reaction buffer additive (prepared in step 4.2)

2.5 µL

5 µL

10 µL

12.5 µL

25 µL

Total Volume

100 µL

200 µL

400 µL

500 µL

1 mL



  1. Incubate coverslips for 30 minutes at room temperature, protected from light.

  2. Remove the Click-iT® reaction cocktail and wash each coverslip with 3% BSA in 1X PBS for 5 minutes.

  3. For antibody staining, proceed to section 5, or for DNA staining, proceed to section 6. If no additional staining is desired, proceed to Imaging and Analysis.


5. Antibody Detection (Optional)

  1. If required, block the coverslips or wells with 3% BSA in 1X PBS for the recommended time, protected from light. Remove the blocking solution.

  2. Prepare and add the primary antibody solution as recommended by the manufacturer.

  3. Incubate for the recommended time and temperature, protected from light. Remove the primary antibody solution.

  4. Wash each coverslip or well twice with 3% BSA in PBS. Remove the wash solution.

  5. Prepare and add the secondary antibody solution as recommended by the manufacturer.

  6. Incubate for the recommended time and temperature, protected from light. Remove the secondary antibody solution.

  7. Wash each coverslip or well twice with 3% BSA in PBS. Remove the wash solution.

For DNA staining, proceed to section 6. If no additional staining is desired, proceed to Imaging and Analysis.




6. DNA Staining

  1. Dilute Hoechst 33342 (Component F) 1:5,000 in PBS to obtain a 1X Hoechst 33342 solution.

  2. Add 100 µL 1X Hoechst 33342 solution per coverslip or well and incubate for 15 minutes at room temperature, protected from light. Remove the Hoechst 33342 solution.

  3. Wash each coverslip or well twice with PBS. Remove the wash solution.


Imaging and Analysis

The Click-iT® TUNEL stained cells are compatible with all methods of slide preparation including wet mount or prepared mounting media. See Table 6 for the approximate fluorescence excitation/emission maxima for Alexa Fluor® dyes and Hoechst 33342 bound to DNA.



Table 6. Approximate Fluorescence Excitation/emission Maxima.

Fluorophore

 Excitation (nm)

Emission (nm)

Alexa Fluor® 488

495

519

Alexa Fluor® 594

590

615

Alexa Fluor® 647

650

670

Hoechst 33342 bound to DNA

350

460


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