One-step TUNEL Apoptosis Detection Kit (Orange, CY3)

I Self-provided reagents

1. Cell samples and frozen sections:

Freshly prepared 4% paraformaldehyde (in PBS), 0.2% Triton X-100 (in PBS). Suspended cells detected by flow cytometry additionally require: 20mM EDTA (pH 8.0), 10mg/mL BSA (in PBS).

These reagents can be prepared 1-2 days before your operation and stored at 4°C.

2. Paraffin sections:

Xylene, anhydrous ethanol

3. Other reagents:

PBS, ddH₂O, Antifade Mounting Medium with DAPI (Cat.No. GC26283)

II Experimental protocol

1. Cell slide samples

1.1. Sample pretreatment

1.1.1. In a well plate of the appropriate size, culture adherent cells on the cell coverslips treated with TC. After apoptosis induction, rinse the coverslips twice with PBS.

1.1.2. Add an excess of 4% paraformaldehyde to completely immerse the coverslips and place at 4°C for 25 minutes to fix the cells.

1.1.3. Wash 2-3 times with PBS solution for 5 minutes each time at room temperature.

1.1.4. Add an excess of 0.2% Triton X-100 and permeabilize at room temperature for 5 minutes.

Note: Triton X-100 is preferred for permeabilization. Proteinase K can also be used for permeabilization. Dilute the 2mg/mL Proteinase K solution with PBS at a ratio of 1:100 to a final concentration of 20μg/mL. Add 100μL of diluted Proteinase K solution to each sample, so that the solution covers the entire sample area, and incubate at room temperature for 5 minutes (the incubation time needs to be explored). The longest permeabilization time should not exceed 15 minutes.

1.1.5. Wash 2-3 times with PBS solution for 5 minutes each time.

 1.2. (Optional step). Positive control.

1.2.1. Prepare a wet cassette by pouring a layer of water at the bottom and laying a clean plastic wrap flatly above water.

1.2.2. Dilute 10×DNase I Buffer to 1×DNase I Buffer using ddH₂O at a ratio of 1:10 for later use.

1.2.3. Add 100μL of 1×DNase I Buffer onto the plastic wrap in the wet cassette. Remove the coverslips from the well plate, and place it cell-side down to cover the 1×DNase I Buffer. Equilibrate at room temperature for 5 minutes.

1.2.4. Dilute DNase I (2000U/mL) with 1×DNase I Buffer to a final concentration of 20U/mL.

1.2.5. Add 50μL 20U/mL DNase I solution onto the plastic wrap in the wet cassette.

1.2.6. Take the coverslips out, gently absorb the excess liquid, and place it cell-side down onto the 20U/mL DNase I solution. Incubate at 37°C for 10 minutes.

1.2.7. Take out the coverslips, place it in a clean well plate with the cell-side upward, and wash it with excess PBS 3 times, 5min each time.

Note: A separate staining jar or well plate must be used for the positive control. The residual DNase I on the positive control may cause false positive signals in the experimental group.

1.3. Marking and detection

1.3.1. Prepare a light-protected wet cassette, pour a layer of water at the bottom, and laying a clean plastic wrap flatly above water.

1.3.2. Dilute 5×Equilibration Buffer to 1×Equilibration Buffer using ddH₂O at a ratio of 1:5.

1.3.3. Add 100μL of 1×Equilibration Buffer to the plastic wrap in the wet cassette, take out the coverslips from the well plate, cover the 1×Equilibration Buffer with cell-side down, and equilibrate at room temperature for 5-10 minutes.

1.3.4. During the equilibration, prepare the TdT labeling solution according to the table below in an ice bath and light-protected conditions.

1.3.5. After equilibration, add 50μL of labeling solution onto clean plastic wrap. Take out the coverslips, gently absorb the excess liquid, and place them cell-side down onto the corresponding labeling solution, taking care to avoid light.

Note: Labeling solution volume: For reactions covering an area less than 5cm², use 50μL per reaction. Determine the total volume of TdT labeling solution needed by multiplying 50μL by the number of experimental and positive control reactions. For larger surface areas, the reagent volume can be increased proportionally.

1.3.6 Close the lid of the wet cassette tightly, avoid light, prevent the coverslips from drying, and incubate at 37℃ for 60min.

1.3.7 Take out the coverslips, gently remove excess liquid and wash 2 times with fresh PBS solution for 5min each room temperature.

Note: If the background is too high, in order to reduce the background, after washing with PBS, the sample can be washed 3 times with PBS containing 0.1% Triton X-100 and 5mg/mL BSA for 5min, so that the free unreacted markers can be removed.

1.3.8. Add one drop of Antifade Mounting Medium with DAPI (Cat. No. GC26283) onto the slide.

1.3.9. Remove the coverslip, gently remove excess liquid, place the coverslip onto the Antifade Mounting Medium with DAPI on the slide, and seal it.

1.3.10 Immediately analyze the sample with a fluorescence microscope, observe the orange fluorescence at the excitation/emission wavelength of 550/570nm; or observe the blue fluorescence of DAPI at the excitation/emission wavelength of 356/451nm.

2. Cell smears samples

2.1. Sample pretreatment

2.1.1. Resuspend the cells in PBS at a concentration of 2×10⁶cells/mL. Pipette 50-100μL of cell suspension onto a poly-lysine-coated slide and allow it to air dry.

2.1.2. Immerse the smear in a staining jar containing 4% paraformaldehyde and incubate at 4°C for 25 minutes to fix the cells.

2.1.3. Wash with PBS solution 2-3 times, each time at room temperature for 5min.

2.1.4. Immerse the slides in 0.2% Triton X-100 solution and incubate at room temperature for 5 minutes to permeabilize the cells.

Note: Triton X-100 is recommended for permeabilization. Proteinase K can also be used for permeabilization. Dilute 2mg/mL Proteinase K solution with PBS at a ratio of 1:100 to a final concentration of 20μg/mL. Apply 100μL of the diluted Proteinase K solution to each sample, ensuring the solution covers the entire sample area. Incubate at room temperature for 5 minutes (Optimizing the incubation time is essential).

2.1.5. Wash with PBS solution 2-3 times, each time at room temperature for 5min.

2.1.6. Carefully use filter paper to absorb the liquid around the sample on the slides. Place the processed sample in a wet cassette to maintain moisture.

2.2. (Optional step). Positive control.

2.2.1. Prepare a light-protected wet cassette by placing wetted tissues at the bottom and transferring all slides into the wet cassette.

2.2.2. Dilute 10×DNase I Buffer to 1×DNase I Buffer using ddH₂O at a ratio of 1:10 for later use.

2.2.3. Add 100μL of 1×DNase I Buffer to the permeabilized samples and equilibrate at room temperature for 5 minutes.

2.2.4. Dilute DNase I (2000U/mL) with 1×DNase I Buffer to a final concentration of 20U/mL.

2.2.5. Gently absorb excess liquid on the glass slide using filter paper. Apply 100μL of the 20U/mL DNase I solution to each positive control sample.

2.2.6. Incubate at 37°C for 10 minutes.

2.2.7. Gently absorb excess liquid on the slides and wash with PBS 3 times, 5 minutes each time.

Note: A separate staining jar or well plate must be used for the positive control. The residual DNase I on the positive control may cause false positive signals in the experimental group.

2.3. Labeling and detection

2.3.1. Place a wetted tissues at the bottom of the wet cassette and transfer all the slides into the wet cassette.

2.3.2. Dilute 5×Equilibration Buffer to 1×Equilibration Buffer with ddH₂O at a ratio of 1:5.

2.3.3. Add 100μL 1×Equilibration Buffer to the sample in the wet cassette and equilibrate at room temperature for 5-10 minutes.

2.3.4. During the equilibration, prepare the labeling solution according to the table below in an ice bath and light-protected conditions.

2.3.5. After equilibration, gently remove excess liquid. Add 50μL of labeling solution to the sample, taking care to avoid light.

Note: Labeling solution volume: For reactions covering an area less than 5cm², use 50μL per reaction. Determine the total volume of TdT labeling solution needed by multiplying 50μL by the number of experimental and positive control reactions. For larger surface areas, the reagent volume can be increased proportionally.

2.3.6 Close the lid of the wet cassette tightly, avoid light, prevent the coverslips from drying, and incubate at 37℃ for 60min.

2.3.7 Take out the coverslips, gently remove excess liquid and wash 2 times with fresh PBS solution for 5min each room temperature.

Note: If the background is too high, in order to reduce the background, after washing with PBS, the sample can be washed 3 times with PBS containing 0.1% Triton X-100 and 5mg/mL BSA for 5min, so that the free unreacted markers can be removed.

2.3.8. Gently absorb excess liquid on the slides, add one drop of Antifade Mounting Medium with DAPI (Cat. No. GC26283) onto the sample.

2.3.9 Cover the Antifade Mounting Medium with DAPI on the slide with a coverslip to seal the slide.

2.3.10 Immediately analyze the sample with a fluorescence microscope, observe the orange fluorescence at the excitation/emission wavelength of 550/570nm; or observe the blue fluorescence of DAPI at the excitation/emission wavelength of 356/451nm.

3. Paraffin-embedded tissue sections

3.1. Sample pretreatment

3.1.1. Immerse the sections in xylene at room temperature, performing the process twice for 5-10 minutes each to completely remove paraffin.

Note: Xylene is toxic and highly volatile, conduct this step in a dedicated laboratory or fume hood. Low temperature may affect the effect of xylene dewaxing. Therefore, the time of xylene dewaxing can be extended to 20min when the room temperature is lower than 20°C.

3.1.2. Immerse the sections in absolute ethanol, soaking and rinsing twice for 5 min each at room temperature.

3.1.3. Immerse the sections in 90%, 80%, and 70% ethanol for 3 min each at room temperature.

3.1.4. Soak and wash the sections with PBS and carefully absorb excess liquid around the sample on the slides with filter paper.

3.1.5 Dilute Proteinase K solution at a concentration of 2mg/mL with PBS at a ratio of 1:100 to a final concentration of 20μg/mL. Add 100μL of diluted Proteinase K solution dropwise to each sample to cover the entire sample area and incubate for 15-30min at room temperature. 

Note: Reaction times may vary depending on the tissue type or species. Overexposure to Proteinase K can cause samples to detach from the slides in subsequent steps, while insufficient permeabilization may lead to low labeling efficiency. A pre-experiment is recommended to optimize reaction conditions.

3.1.6. Immerse and wash the samples 3 times with PBS solution for 5min each. Gently remove excess liquid and carefully absorb the liquid around the sample on the slide with filter paper. The treated sample is kept moist in a wet cassette.

Note: Ensure that Proteinase K is thoroughly removed during this step, as any residual enzyme can significantly interfere with subsequent labeling reactions.

3.2. Positive Control (Optional Step)

Refer to the procedures described for positive control in 2.2.

3.3. Labeling and Detection

Refer the labeling and detection steps described for cell smear samples in 2.3.

4. Frozen tissue sections

4.1. Sample pretreatment

4.1.1. Place the frozen tissue sections on a long rack and dry at room temperature for 20 min.

4.1.2. Immerse the sections in 4% paraformaldehyde solution and fix at room temperature for 30min.

4.1.3. Gently absorb excess liquid on the slides with filter paper.

4.1.4. Immerse the sections in PBS solution and wash twice for 5min each. Gently absorb excess liquid on the slides with filter paper.

4.1.5. Drop 100μL of 0.2% Triton X-100 to cover the entire sample area each, permeabilize at room temperature for 15-30 minutes. If the permeabilization is not sufficient, dilute Proteinase K solution with PBS at a concentration of 2mg/mL at a final concentration of 20μg/ml at a ratio of 1:100. Drop 100μL of diluted Proteinase K solution to each sample so that the solution covers the entire sample area and incubate for 10-30min at room temperature.

Note: Incubation times may vary depending on the tissue type or species. Overexposure to Proteinase K may cause samples detached from the slides during subsequent steps, while insufficient permeabilization may lead to low labeling efficiency. A pre-experiment is recommended to optimize reaction conditions.

4.1.6. Immerse and wash the samples 3 times with PBS solution for 5min each. Gently remove excess liquid and carefully absorb the liquid around the sample on the slide with filter paper. The treated sample is kept moist in a wet cassette.

Note: Ensure that Proteinase K is thoroughly removed during this step, as any residual enzyme can significantly interfere with subsequent labeling reactions.

5. Suspension cells detected by flow cytometry

5.1. Wash (3-5)×10⁶ cells twice with PBS, centrifuge at 4℃ 1800rpm (300×g) for 5min each time. Then resuspend with 0.5mL PBS.

Note: The cells required for detection with this kit is not less than 5×10⁵.

5.2. Add 5mL 4% paraformaldehyde solution and incubate at 4℃ for 40min to fix the cells.

Note: To prevent cells from congregating into clumps, fixation can be performed while shaking slowly on a shaker, or mix well every 10min.

5.3. Centrifuge at 4℃ at 600×g for 5min, discard the supernatant and resuspend the cells with 10 mg/mL BSA in PBS.

5.4. Repeat the centrifugation and resuspend the cells with 0.2mL 10 mg/mL BSA in PBS.

5.5. Add 2mL of 0.2% Triton X-100 solution and incubate for 5min on ice.

Note: The refractive index of cells will decrease after permeabilization, and the cells will be difficult to observe. Handle carefully to avoid cell loss during the whole process.

5.6. Add 10mL 10mg/mL BSA in PBS, centrifuge at 4℃ at 600×g for 5min, discard the supernatant and resuspend the cells with 5mL 10mg/mL BSA in PBS. Repeat the centrifugation and resuspend the cells with 1mL 10mg/mL BSA in PBS.

5.7. Transfer 2×10⁶ cells to a new 1.5mL centrifuge tube.

5.8. Centrifuge at 600×g for 5min at 4°C, discard the supernatant, and resuspend the cells with 100μL 1×Equilibration Buffer (dilute 5×Equilibration Buffer in a 1:5 ratio with ddH2O). Incubate at room temperature for 5min.

5.9. During the equilibration, prepare the labeling solution according to the table below in an ice bath and light-protected conditions.

5.10. Centrifuge at 600×g for 5min at 4°C, discard the supernatant, and resuspend the cells with 50μL labeling solution. Incubate at 37°C for 60min in the dark. Flick the tube wall every 15 minutes or gently resuspend the cells with a micropipette.

Note: the labeling volume is 50μL for each standard reaction of 2×10⁶ cells. Multiply 50μL by the number of reactions to determine the total volume of TdT labeling reaction solution required.

5.11. Add 1mL of 20mM EDTA to terminate the reaction and mix gently with pipette.

5.12. Centrifuge at 600×g for 5min at 4°C, discard the supernatant, and resuspend the cells with 10 mg/mL BSA in PBS. Repeat centrifuge and resuspend once.

Note: PBS (containing 10mg/mL BSA) buffer is used to reduce cell loss caused by centrifugation.

5.13. Analyze cells using flow cytometry. Select CY3 channel for detection (Ex/Em: 550/570nm)

Note: Please detect as soon as possible to avoid fluorescence quenching.

III Cautions

1. 5×Equilibration Buffer contains surfactant, so it will diffuse and drain when added to the slide of cell smear/section sample. It is recommended to circle the staining area with PAP Pen before the experiment.

2. Dissolve 5×Equilibration Buffer at room temperature before your experiment. It's a normal phenomenon that 5×Equilibration Buffer crystallize after melting. Vortex thoroughly before use.

3. Avoid vortexing and repeated freeze-thaw cycles for both CY3-dUTP Mix and TdT Enzyme. Before use, place CY3-dUTP Mix on ice to dissolve completely. Once dissolved, centrifuge and pipette to mix. TdT Enzyme is temperature-sensitive. Please store it strictly at -20°C, take it out before use, and put it back immediately after use.

4. When preparing the labeling solution and the positive control DNase I solution, it is recommended not to vortex.

5. When performing Tunel detection by flow cytometer, avoid cell damage and excessive bubbles when using a pipette to blow cells. If the sample is adherent cells, some apoptotic cells will be suspended in the supernatant, so the cells in the supernatant should also be collected.

6. Please keep the samples moist during the experiment, as drying could cause experimental failure.

7. This kit is for research use only.

8. Please take safety precautions and follow the procedures of laboratory reagent operation.

9. The conditions recommended in this manual are universal. Users can optimize the sample processing time, reagent concentration and other conditions according to different sample types and pre-experiment results, and select the most suitable experimental conditions.

IV Troubleshooting