CHAPTER 3 MATERIALS AND METHODS
3.9 TUNEL assay
3.9.1 Principle
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay was first developed in 1992 by Gavrieli et al. This method detects cells undergoing programmed cell death (PCD) in situ at the single cell level without destroying cell and tissue architecture. Apoptosis is associated with the activity of endogenous endonucleases which cleave double stranded genomic DNA at regular intervals and yield numerous 3’hydroxyls (3’-OH) available for TdT to label (Bortner et al. 1995; Didenko and Hornsby, 1996). In the TUNEL assay, cells are treated with protease to expose the
nuclear DNA. Thereafter TdT is added which binds to the 3’-OH ends of the cleaved DNA and facilitates the incorporation of biotinylated deoxyuridine (dUTP). If this is the case, horseradish peroxidase (HRP)-labeled streptavidin is bound to dUTP. These biotinylated nucleotides are detected using hydrogen peroxide, the substrate for HRP, and diaminobenzidine (DAB), which is a stable chromagen detectable by light microscopy.
3.9.2 Methodology
HaCaT or ME-180 cells were grown on glass coverslips in 24-well tissue culture plates for 2 days until confluent. The residual CGM-E was removed and replaced with 500µl CGM- E, then infected with C. trachomatis serovar L2, L3, E, or the clinical isolates US151, US162 and US197 in 100 µl SPG at an MOI of 0.25. One hundred microlitres sterile SPG was used for the uninfected control, TUNEL positive control, and TUNEL negative control (provided with the kit). Monolayers were centrifuged at 1200 x g for 1 hour, then incubated at 37°C or 33°C for 1 hour. The residual inoculum was aspirated, replaced with 1ml CGM-E and the monolayers incubated at 37°C or 33°C for 2 or 5 days.
After 2 days (or 5 days for the HaCaT cells at 33°C), the cells were fixed and stained for the TUNEL assay using the DeadEndTM Colorimetric TUNEL System (Promega) and C. trachomatis detection using the MicroTrak® Chlamydia trachomatis Culture Confirmation Kit (Trinity Biotech) as described below.
Procedures were carried out according to manufacturer’s instructions with the following adaptations:
• Cells were processed on coverslips within the wells of a 24 well plate and mounted to glass slides after the staining procedure, instead of being processed on glass slides
• Cells were fixed with 4 % formalin in PBS in stead of 10% buffered formalin, 4%
paraformaldehyde solution or 10 % buffered formalin in PBS
• Cells were permeabilized with 0.4% Triton® X-100 in PBS for 10 min at room temperature for ME-180 cells, and 37°C for HaCaT cells. The manufacturer’s recommendation of 0.2% Triton® X-100 in PBS for 5 min did not permeabilize the HaCaT cells sufficiently.
• Ten units per millilitre RQ1 DNase was used as the TUNEL positive control
• At the end of the TUNEL assay staining procedure, the same slides were stained with the MicroTrak® C. trachomatis Culture Confirmation Test Kit
Cells were processed directly in the wells at room temperature unless otherwise stated.
Procedure:
1. Wash with PBS
2. Fix with 4% formalin in PBS for 25 min 3. Wash twice with PBS for 5 min
4. Permeabilize with 0.4% Triton® X-100 in PBS for 10 min (room temperature for ME- 180 cells; 37°C for HaCaT cells)
5. Wash twice with PBS for 5 min
6. DNase treatment for TUNEL positive control:
a. Wash with 200µl DNase I buffer for 5 min
b. Treat with 200µl RQ1 RNase free DNase (10 unit/ml) (Promega) in DNase I buffer for 10 min at 37°C
c. Rinse with tdH2O 4 times d. Wash with PBS for 5 min
7. Equilibrate with 100µl equilibration buffer for 5-10 minutes
8. Prepare rTdT reaction mix by combining the following components per well:
98µl equilibration buffer 1µl biotinylated nucleotide mix
1µl rTdT enzyme or 1µl autoclaved tdH2O for the TUNEL negative control
9. Label with 100µl rTdT reaction mix for 60 min at 37°C with each glass coverslip covered with a plastic coverslip to ensure even distribution of the reagent and prevent evaporation
10. The plastic coverslips were removed and the reaction stopped with 200µl 2X SSC for 15 min
11. Wash thrice with PBS for 5 min
12. Block endogenous peroxidases with 0.3% hydrogen peroxide for 4 min 13. Wash thrice with PBS for 5 min
14. Bind with 200 µl streptavidin horse radish peroxidase (HRP) (1:500 in PBS) for 30 min 15. Wash thrice with PBS for 5 min
16. Prepare DAB stain solution:
950 µl tdH2O
50 µl 20x DAB substrate buffer 50 µl DAB 20x chromagen 50 µl 20x hydrogen peroxide
17. Stain with 100µl DAB solution for 15 min with each glass coverslip covered with a plastic coverslip to ensure even distribution of the reagent and prevent evaporation 18. Remove plastic coverslips
19. Rinse monolayer 5 times with tdH2O
20. Counterstain for C. trachomatis detection using the MicroTrak® Chlamydia trachomatis Culture Confirmation Kit (Trinity Biotech)
a. Moisten with PBS for 5 min
b. Stain with 30µl stain reagent for 30 min at 37°C in a humidified chamber c. Wash with tdH2O for 10 sec
21. Remove glass coverslip from well 22. Blot
23. Mount to glass slide with DPX mounting fluid
Once the mounting fluid had solidified, the slides were viewed with a Nikon eclipse E600 fluorescent microscope. Slides were examined using bright field microscopy to identify cells with fragmented DNA and DAB positive nuclei; then switched to fluorescence (excitation wavelength 450-490 nm; emission wavelength 520 nm) and the same field of view examined for the presence and location of C. trachomatis-infected cells relative to DAB positive cells.
Images were captured in colour using a Nikon ColorView Soft Imaging System digital camera. For each field of view, DAB positive cells were captured in a bright field photograph, and the same field of view captured under fluorescence. Each pair of images was examined to identify relationships between DAB positive and C. trachomatis infected cells.