Abstract
Male infertility affects men worldwide with about 20 percent of couples having male factor infertility [1]. The routine semen analysis is the first step in the assessment of male infertility. However, conventional semen analysis does not provide a complete understanding of fertility potential, especially in patients with idiopathic infertility [2]. In this scenario, DNA integrity is the most important feature to ensure normal fertilization, implantation, pregnancy and embryonic development. Sperm DNA fragmentation (SDF) can be due to several intrinsic factors such as varicocele, oxidative stress, apoptosis, and chromatin packaging defects. Further, SDF can be caused by extrinsic factors such as lifestyle alterations, infections, exposure to xenobiotics, etc. [3–7].
19.1 Introduction
Male infertility affects men worldwide with about 20 percent of couples having male factor infertility [1]. The routine semen analysis is the first step in the assessment of male infertility. However, conventional semen analysis does not provide a complete understanding of fertility potential, especially in patients with idiopathic infertility [2]. In this scenario, DNA integrity is the most important feature to ensure normal fertilization, implantation, pregnancy and embryonic development. Sperm DNA fragmentation (SDF) can be due to several intrinsic factors such as varicocele, oxidative stress, apoptosis, and chromatin packaging defects. Further, SDF can be caused by extrinsic factors such as lifestyle alterations, infections, exposure to xenobiotics, etc. [3–7].
Sperm nuclear DNA is highly compacted because of its protamine content. DNA fragmentation can be attributed to several factors such as oxidative stress [4–6], abortive apoptosis [5], failure to repair DNA strand breaks [6], and environmental exposure of sperm DNA to toxins, defective chromatin packaging and protamine deficiency [7]. Sperm DNA damage results in infertility, miscarriage, and birth defects in offspring [8]. Oxidative stress is the main cause of sperm DNA damage. Oxidative stress is caused by an imbalance between the levels of oxidants or reactive oxygen species (ROS) and the ability of the antioxidants or reductants to scavenge them. A number of factors can lead to oxidative stress, including (viral or bacterial) infections, exposure to xenobiotics, tobacco and alcohol consumption, consumption of fatty diet, drug abuse, radiation, psychological stress, consumption of medications (cyclophosphamide, opioids, etc.), exposure to environmental and air pollutions, chronic diseases, cryptorchidism, and testicular torsion [3, 9–12].
19.2 Mechanisms of Sperm DNA Damage
DNA fragmentation may occur during spermiogenesis where DNA is condensed as a result of replacement of histones with protamines and packaged into the differentiating sperm head as a result of the nuclear exchange of proteins (transition proteins and protamines). This supercoiling of the nucleosomal DNA can result in torsional stress. Endogenous endonucleases (topoisomerases) may induce DNA fragmentation to counter this stress [13, 14]. Although spermatozoa are transcriptionally and translationally inactive and cannot undergo conventional programed cell death or “regulated cell death” called “apoptosis”, they exhibit some of the hallmarks of apoptosis. This includes caspase activation and phosphatidylserine exposure on the surface of the sperm. This process is termed “abortive apoptosis” [5, 15]. During spermatogenesis, sperm cells have the ability to repair some DNA damage, however this innate ability is lost once they mature [16, 17]. Therefore, post-testicular sperm are more vulnerable to DNA damage [18–21]. Studies have shown that the accurate assessment of sperm DNA integrity expressed as SDF is a good predictor of semen quality [22–25]. Many tests have been developed to measure SDF, but the most commonly used tests are terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay, Sperm Chromatin Structure Assay (SCSA®), Comet and Sperm Chromatin Dispersion (SCD) assay [26]. The tests that assess SDF can be classified as direct and indirect tests. Direct tests include TUNEL and Comet assay, and they measure single or double strand damage. SCSA and SCD are the indirect tests that measure the susceptibility of sperm DNA breaks after acid or heat denaturation (Table 19.1).
| Assay | Principle | Parameter measured |
|---|---|---|
| Direct assays | ||
| TUNEL | Adds labeled nucleotides to free DNA ends | % Cells with labeled DNA |
| Template independent | ||
| Labels SS and DS breaks | ||
| COMET | Electrophoresis of single sperm cells | % Sperm with long tails (tail length, % of DNA in tail) |
| DNA fragments form tail | ||
| Intact DNA stays in head | ||
| Alkaline COMET | ||
| Alkaline conditions, denatures all DNA | ||
| Identifies both DS and SS breaks | ||
| Neutral COMET | ||
| Does not denature DNA | ||
| Identifies DS breaks, maybe some SS breaks | ||
| In situ nick translation | Incorporates biotinylated dUTP at SS DNA breaks with DNA polymerase I | % Cells with incorporated dUTP (fluorescent cells) |
| Template-dependent | ||
| Labels SS breaks, not DS breaks | ||
| Indirect assays | ||
| DNA break detection FISH | Denatures nicked DNA | Amount of fluorescence proportional to number of DNA breaks |
| Whole genome probes bind to SS DNA | ||
| SCD | Individual cells immersed in agarose | % Sperm with small or absent halos |
| Denatured with acid then lysed | ||
| Normal sperm produce halo | ||
| Acridine orange flow cytometric assays (example: SCSA) | Mild acid treatment denatures DNA with SS or DS breaks | DFI – the percentage of sperm with a ratio of red to (red + green) fluorescence greater than the main cell population |
| Acridine orange binds to DNA | ||
| DS DNA (nondenatured) fluoresces green | ||
| SS DNA (denatured) fluoresces red | ||
| Flow cytometry counts thousands of cells | ||
| Acridine orange test | Same as above, manual counting of green and red cells | % Cells with red fluorescence |
Each of these tests is related to properties of the DNA damage and provides semi-quantitative estimates only. They do not provide information of the specific DNA sequences that may be affected [27]. High DNA integrity is generally observed in fertile men with normal semen parameters, whereas infertile men with abnormal semen parameters have higher percentage of DNA damage. However, men with normal semen parameters can have poor DNA integrity [28, 29]. There is, however, some evidence to suggest that increased DNA fragmentation is associated with reduced fertility [30]. Yet, this evidence is not conclusive for these tests to be truly predictive of fertility status.
Five percent of women experience two consecutive miscarriages and approximately 1 percent have three or more consecutive miscarriages [31–33]. This is linked with sperm DNA damage [34, 35].
A panel of experts in the reproductive field extensively analyzed the utility of SDF as part of the male fertility evaluation [2]. They recommended SDF analysis for men with high grade varicocele and normal semen parameters as well as low grade varicocele and abnormal semen parameters [2]. Other conditions include unexplained infertility, recurrent pregnancy loss, and recurrent intrauterine insemination (IUI) failures [2]. Nevertheless, many reproductive societies such as the American Society for Reproductive Medicine (ASRM), European Association of Urology (EAU), American Urological Association (AUA) and National Institute of Clinical Excellence (NICE) do not recommend its use as part of the routine assessment of male infertility [25, 26].
In this chapter, we summarize the step-by-step protocols established for the measurement of SDF in human spermatozoa using the bench top flow cytometer. Data acquisition and analysis as well as the necessity for quality control evaluation is further discussed. These steps are required for the standardization of laboratory protocols and the establishment of reference values for TUNEL assay applicable to their patient population.
19.3 Assays for the Evaluation of Sperm DNA Damage
Sperm DNA damage can be assessed by a number of techniques that measure different aspects of DNA damage (Table 19.1). Each assay has its own advantages and disadvantages (Table 19.2). One of the most commonly used assays is the TUNEL assay. TUNEL identifies what is termed as “real” DNA damage – that is, damage that has already occurred – as opposed to “potential” damage caused by exposing sperm to denaturing conditions tested by indirect tests (Table 19.3).
| Direct Assays | Advantages | Disadvantages |
|---|---|---|
| TUNEL | Can be performed on few sperms | Thresholds not standardized |
| Expensive equipment not required | Variable assay protocols | |
| Simple and fast | Not specific to oxidative damage | |
| High sensitivity | Special equipment required (flow cytometer) | |
| Indicative of apoptosis | ||
| Correlated with semen parameters | ||
| Associated with fertility | ||
| Available in commercial kits | ||
| Comet | High sensitivity | Labor intensive |
| Simple and inexpensive | Not specific to oxidative damage | |
| Correlates with seminal parameters | Subjectiveness in data acquired | |
| Small number of cells required | No evident correlation in fertility | |
| Lack of standard protocols | ||
| Can perform on few sperm | Requires imaging software | |
| Alkaline: identifies all breaks | Variable assay protocols | |
| Neutral: may identify more clinically relevant breaks | Alkaline: may identify clinically unimportant fragmentation | |
| May induce breaks at “alkaline-labile” sites | ||
| In situ nick translation | Simple | Unclear thresholds |
| Indirect assays | Less sensitive | |
| DNA break detection FISH | Can perform on few sperm | Limited clinical data |
| SCD | Easy, can use bright-field microscopy | Limited clinical data |
| Acridine orange flow cytometric assays (SCSA) | Many cells rapidly examined | Expensive equipment required |
| Most published studies reproducible | Small variations in lab conditions affect results | |
| Calculations involve qualitative decisions | ||
| Manual acridine orange test | Simple | Difficulty with indistinct colors, rapid fading, heterogeneous staining |
| 8-OHdG analysis | High specificity | Large amount of sample required |
| Quantitative | Introduction of artifacts | |
| High sensitivity | Special equipment required | |
| Correlated with sperm function | Lack of standard protocols | |
| Associated with fertility |
| TUNEL | SCSA | COMET | SCD | |
|---|---|---|---|---|
| Principal | 1. Adds labeled nucleotides to free DNA ends 2. Template independent 3. Labels SS and DS breaks | 1. Mild acid treatment denatures DNA with SS or DS breaks 2. Acridine orange binds to DNA 3. Double stranded DNA (nondenatured) fluoresces green. Single stranded DNA (denatured) fluoresces red 4. Flow cytometry counts thousands of cells. | 1. Electrophoresis of single sperm cells 2. DNA fragments form tail 3. Intact DNA stays in head Alkaline COMET 1. Alkaline conditions, denatures all DNA. 2. Identifies both DS and SS breaks Neutral COMET 1. Does not denature DNA 2. Identifies DS breaks, maybe some SS breaks. | Individual cells are immersed in agarose. Cells are denatured and lysed A distinct halo is seen in spermatozoa with intact DNA integrity. |
| What is measured | % Cells with labeled DNA | DFI – the percentage of sperm with a ratio of red to (red + green) fluorescence greater than the main cell population | % Sperm with long tails (tail length, % of DNA in tail) | Percentage of sperm with small or absent halo The distinct halo of deprotenized nuclei (nucleoids) is measured by bright field or fluorescent microscopy. |
| Type of Assay | Direct | Indirect | Direct | Indirect |
| Objective | Objective | Subjective | Subjective | |
| Ease of assay | Many labs run this assay | Samples have to be shipped to reference lab. | Very few labs perform this assay. | Simple, fast and reproducible |
| Instrumentation | Flow cytometry | Flow cytometry | Microscopy | Microscopy |
| Nature of assay | TUNEL kit available | Only in reference or designated labs. | Manual, no assay kits available | Manual and Halosperm kit available |
| Reference values | Ranges from 10–30% | Robust, >30% DFI indicative of decreased pregnancies | Clinically useful reference values not established | Limited clinical data |
| Type of samples | Fresh or frozen | Fresh or frozen | Fresh | Fresh |
| Repeatability of assay | Good | Good | Poor | |
| Cost | Inexpensive | Expensive | Inexpensive |
All of the assays shown in Table 19.1 strongly correlate with each other. Unfortunately, none is able to selectively differentiate clinically important from clinically insignificant DNA fragmentation. Moreover, DNA nicks that occur physiologically or pathologically cannot be differentiated with these assays, which cannot evaluate the genes that may be affected by DNA fragmentation. All the assays, including TUNEL, can only determine the amount of SDF that occurs with the assumption that higher level of DNA fragmentation is pathological.
19.4 Measurement of DNA Damage in Spermatozoa by TUNEL Assay
DNA damage can be measured using the TUNEL assay by various protocols such as: fluorescein isothiocynate (FITC) labeled dUTP system (In Situ Cell Detection kit, Roche Diagnostics, Indianapolis, IN); biotin-d(UTP)/avidin system; BrdUTP/anti-Br-dUTP-FITC system and the Apoptosis detection kit (Apo-Direct kit; BD Pharmingen, San Diego, CA)
Here we will briefly describe these protocols and elaborate on the step-by-step measurement of SDF by TUNEL test by a bench top flow cytometer using the Apo-Direct kit. In addition, we will describe the detection of SDF using TUNEL kit and fluorescence microscopy.
19.4.1 Principle of TUNEL Assay
The TUNEL assay was first developed for somatic cells and then later adapted for sperm cells. It shows the percentage of apoptotic cells with damaged DNA. Apoptosis in spermatozoa results in activation of endonucleases, enzymes that induce sperm DNA fragmentation [40]. During apoptosis, endonucleases break down high order sperm chromatin into smaller DNA fragments of ~50kb. These breaks are labeled by FITC-dUTP. This is accomplished with the template independent enzyme called terminal deoxyribonucleotidyl transferase (TdT) that transfers the deoxyribonucleotides to the 3-hydroxyl (3-OH) end of the single- and double-strand breaks [1, 36–38] (Figure 19.1). The intensity of fluorescent labeling examined by fluorescence microscope or flow cytometry is proportional to the number of DNA strand break sites.
Figure 19.1 Set-up of a benchtop flow cytometer.
19.4.1.1 In Situ Cell Detection Kit to Detect DNA Fragmentation
The (FITC dUTP system or the In Situ Cell Detection kit (Roche Diagnostics) is used to detect SDF. Spermatozoa are centrifuged (at 600 × g for four minutes) before being resuspended in 100 μL of fresh permeabilization solution (10 mg sodium citrate, 10 μL Triton X‐100 in 10 mL dH2O) and incubated for two minutes at 4°C. The cells are centrifuged again (600 × g for four minutes) and the pellet is washed with PBS. The positive control samples are treated with 100 μL of DNase I provided by the kit (1 mg/mL) supplemented with 10 μL MgSO4 (100 mM) for one hour at 37°C. Cells are washed twice in PBS, diluted to a final volume of 500 μL in PBS and kept in the dark for analysis via flow cytometry.
19.4.1.2 Evaluation by Apo-BrdUTP Apoptosis Detection Kit
Apo-BrdUTP in situ DNA fragmentation kit is also used to measure SDF. The kit includes the positive and the negative controls. The DNA labeling is done with BrdUTP which binds to the 3’-OH terminals of the DNA strand breaks and terminal deoxynucleotidyl transferase (TDT). The antibodies to the BrdUTP molecule are linked to fluorescein molecule. These are detected in FL1 channel [39, 40]. Sample analysis is done with flow cytometer. FL3 channel records the propidium iodide fluorescence.
19.4.1.3 Measurement of DNA Fragmentation by TUNEL Assay Using a Bench Top Flow Cytometer and Apo-Direct Assay Kit
The TUNEL assay can be evaluated by the Bench top Accuri C6 flow cytometer (BD Pharmingen) (Figure 19.2) [1, 36]. Propidium Iodide (PI) is used as a fluorescent counterstain that allows to count all the other intact cells. The TUNEL kit utilized in the following procedure is the APO-DIRECT™ kit (BD Biosciences Pharmingen, San Diego, CA).
A minimum of 10,000 events are examined for each measurement at a flow rate of about 100 events/second on the flow cytometer. The excitation wavelength is 488 nm supplied by an argon laser at 15 mW. Green fluorescence (480–530 nm) is measured in the FL-1 channel and red fluorescence (580–630 nm) in the FL-2 channel. Spermatozoa obtained in the plots are gated using a forward-angle light scatter (FSC) and a side-angle light scatter (SSC) dot plot to gate out debris, aggregates and other cells different from spermatozoa [1, 36].
19.4.1.3.1 Protocol
19.4.1.3.1.1 Materials
– Reagent solutions used, equipment, test specimens
A. Sheath fluid (blue bottle): it comprises of 0.22 µm filtered, deionized water with or without bacteriostatic concentrate solution (PN 653156).
If bacteriostatic concentrate solution is used (optional), add one bottle per 1 L of water.
B. Cleaning solution (green bottle) (PN 653157): cleaning concentrate solution. To dilute add 3 mL of cleaning concentrate to 197 mL of filtered deionized water. Use the solution within two weeks.
C. Decontamination solution (yellow bottle) (PN653154):
Add entire bottle to 180 mL of filtered, deionized water.
D. Extended flow cell clean (PN 653159): this solution is provided in working concentrate.
E. APO-DIRECT™ Kit (BD Pharmingen, Catalog #556381)
1. PI/RNase staining buffer
2. Reaction buffer
3. FITC-dUTP
4. TdT Enzyme
5. Rinsing buffer
6. Wash buffer
F. Serological pipettes (2 mL and 5 mL)
G. Eppendorf pipette and tips (20 µL, 100 μL and 1000 μL)
H. Sperm counting chamber
I. Paraformaldehyde (3.7 percent)
19.4.1.4 General Set-Up of the Bench Top Cytometer
A. Open the software.
B. Inspect all the reagent bottles to ensure that the fluid levels are fine.
C. Waste bottle should be empty.
D. The sheath, cleaner and the decontamination bottles must be full.
E. Turn on the cytometer.
F. At the beginning, the software light turns yellow. This is an indication that the peristaltic pump has started to run.
G. Allow five minutes for the fluidics line to get flushed with the sheath fluid.
H. Wait for the cytometer software light to turn green, indicating that the C6 Accuri is connected and ready.
I. Flush the tubing to remove any bubbles from the cytometer system.
J. Place a 0.22 µm deionized (DI) water tube on the sheath injection port (SIP).
K. Run a cycle with criteria selected as “Run with limits”.
L. Select “Fluidics speed” as “Fast”.
M. Click the “Run” button.
N. Leave the SIP tube on the tube holder. Save the file as “Flush”.
19.4.1.5 Instrument Quality Control
The quality control is performed with eight-peak beads. The eight-peak beads are 3.2 µm particles excited by the blue laser and emitting light at eight different wavelengths. The validation of the bench top flow cytometer is done by running the eight-peak beads and determining the coefficient of variation (CV) and mean fluorescence intensity (MFI) each time the instrument is used. These can be plotted as CV and MFI in the Levy Jennings chart.
19.4.1.6 Preparation of Eight-Peak Beads
A. Use a 12 × 75 mm tube and label it as “Eight-Peak QC Beads”. Also mark the date of preparation.
B. Add 1 mL of deionized DI water to each of the tubes.
C. Vortex each of the bead vials provided by manufacturer.
D. Place four drops of eight-peak beads to the tube and vortex. Cover the tube with the aluminum foil.
19.4.1.6.1 Preparation for the Run of the Eight-Peak QC Beads
A. Double click and open the eight-peak bead template provided with the instrument (Figure 19.3).
B. Turn on the cytometer by pressing the power button.
C. A green light will be displayed under the “Collect tab” indicating that the machine is ready for sample acquisition.
D. Start the acquisition by clicking on the well “A1”.
E. Place a tube with 2 mL of 0.22 µm-DI water on the SIP.
F. Check “Run with limits” and set the time limit to “15 minutes”.
G. Set “Fluidics” speed to “Fast”.
H. Click the “Run” button.
I. The software will prompt to “Save” the file.
J. After completion of the “Run” place the tube with DI on the SIP.
Figure 19.3 Eight-peak quality control beads as seen after analysis in software; the CV of the brightest peak (M3, M6, M9) is measured.
19.4.1.6.2 Acquisition of the Eight-Peak Bead Data
A. Select an empty field from left heading towards the right selecting one well at a time from A1 to H12.
B. Enter in the empty space above the wells the acquisition date for the eight-peak beads as “eight-peak-beads-date-tech initials”.
C. The acquisition is performed under the “Collect tab”.
D. Unselect the “Time” check-box next to “Min.” and “Sec.”.
E. Select the “Events” check-box and check the “50,000” option in the “Events” field.
F. From the drop-down menu click on “Ungated sample”.
G. Set “Fluidics” speed to “Slow.”
H. Mix the eight-peak QC bead suspension by vortexing the tube.
I. Remove tube of deionized water from the SIP.
J. Place the “Eight-Peak QC Bead” tube under the SIP.
K. Click the “RUN” button to start the acquisition.
L. Save the file as “Eight-Peak QC-DATE-TECH INITIALS”.
M. After the cytometer has recorded 50,000 events, acquisition will stop.
N. When the run is finished, remove “Eight-Peak QC Bead” tube from SIP and clean the SIP using a lint-free wipe.
O. Place the tube containing 2 mL of DI water on the SIP.
19.4.1.6.3 Ending the Run
A. Place a 2 mL tube of DI water on the SIP and select an empty well in the BD Accuri software.
B. Check “Time” and set the time to “2 min.”.
C. Set “Fluidics” speed to “Fast.”
D. Click the “Run” button.
E. When the run is finished, place the tube with 2 mL of DI water on the SIP.
F. Before running any other samples click “delete events” to erase the data collection from the water run.
G. At the completion off the run, use a 10 percent solution of bleach for two minutes, followed by a DI water run before shutting down the instrument.
19.4.1.6.4 Analyzing the Eight-Peak Bead Acquisition Data
A. The analysis is done in the “Collect” tab only.
B. Select the well (example: well A1) where the data was acquired for the eight-peak beads run.
C. Adjust the R1 gate to include 75–85 percent of all events.
D. The first plot is labeled “FSC-H” and “SSC-H”, click on the border of the “R1” gate. The border will become bold and handles will appear to adjust the gate settings.
E. Include all the “Singlets” or the main bead population making sure to exclude all the doublets which appear as light-gray dots.
F. All the three plots “FL1-H”, “FL2-H” and “FL3-H” must be gated on R1.
G. Measure the CV of the brightest peak (right most peak) of the “FL1-H”, “FL2-H” and “FL3-H” histograms (Figure 19.3).
19.4.1.6.5 Criteria for Successful Eight-Peak Bead Quality Control
The CV for all three peaks must be less than 5 percent for validation of the three channels of the instrument.
A. To select the brightest peak, use the zoom tool over the histogram and zoom in on the brightest peak in the “FL1-H” histogram.
B. The “M1” marker is adjusted tightly around the brightest peak.
C. The above two steps need to be repeated around the “FL2-H” and “FL3-H” histograms as well.
D. Save this template for future runs of the eight-peak quality control.
19.4.1.6.6 Tracking Performance of the Eight-Peak Bead Quality Control
Open the file for the acquisition data obtained from the eight-peak bead run. Highlight all the statistics that need to be copied and transferred to the excel spread sheet. In the “statistics column selector”, check the boxes for the mean and CV of the brightest peak (M3, M6, and M9) for the following parameters: “FL1-H”, “FL2-H” and “FL3-H”. The Levy-Jennings chart gets populated by the data and the data is saved.
19.4.2 Sample Preparation for TUNEL Assay
A. Semen sample is kept in the incubator for 30–60 minutes at 37oC to undergo liquefaction.
B. After liquefaction, sample is evaluated for volume, sperm concentration, total sperm count, sperm motility and round cell concentration.
C. The sample volume for TUNEL needs to be adjusted to 2.5 × 106/mL.
D. This can be achieved by the following formula:
2.5×1000µLSperm Concentration106/mL=XμL
E. Save two tubes each for the test sample, negative control and positive control samples.
F. Label tubes with the following information:
a. TUNEL
b. Patient name
c. Medical record number
d. Date
G. Aliquot the required volume for an adjusted sperm concentration of 2.5 × 106/mL cells to each of the four tubes.
H. Spin the aliquoted sample at 300 × g for seven minutes.
I. Remove the supernatant after the spin.
J. Replace the supernatant with 1 mL PBS.
K. Centrifuge at 300 × g for seven minutes.
L. Remove the supernatant and replace with 1 mL PBS.
19.4.2.1 Preparation of the “Positive Control” Sample
A. Add 100 µL of the stock hydrogen peroxide of 37 percent solution to 1400 µL of PBS 1× to prepare a diluted solution (1:15 dilution) of H2O2.
B. Add and suspend the sperm cells in 1 mL of diluted H2O2 solution.
C. Place the sperm cell resuspended in H2O2 on the heating block at 50°C for 60 minutes.
D. After incubation, centrifuge the tube for seven minutes at 300 × g.
E. Aspirate the supernatant with a transfer pipette, resuspend in 1 mL PBS 1× and centrifuge at 300 × g for seven minutes.
F. Remove the supernatant and replace with 1 mL PBS.
Along with the “Test” “Negative” sample tubes, repeat centrifugation step for seven minutes at 300 × g.
G. Remove the supernatant and replace with 1 mL PBS.
H. Proceed to the next steps of “Fixation” and “Permeabilization.”
19.4.2.2 Fixation and Permeabilization
A. Fixation of the sperm cells is done with paraformaldehyde.
B. The supernatant from the “Test”, “Negative” and “Positive” control sample is removed after centrifugation at 300 × g for seven minutes, followed by addition of 1 mL of 3.7 percent paraformaldehyde solution.
C. Incubate the samples by resuspending in 3.7 percent paraformaldehyde at room temperature for 15 minutes.
D. Centrifuge the samples at 300 × g for four minutes.
E. Carefully aspirate the paraformaldehyde and replace it with 1 mL of PBS.
F. Centrifuge at 300 × g for four minutes.
G. Aspirate the supernatant and replace with 1 mL of ice-cold ethanol (70 percent). Place the sample at 4oC for 15–30 minutes.
19.4.2.3 Staining Protocol
The negative kit control and positive kit controls are provided as part of kit components.
A. The “Negative” controls, “Positive” controls, and “Test” samples should be mixed well by vortexing them.
B. Aliquot 2 mL suspensions of the well mixed “Kit control” samples into 12 × 75 mm polystyrene tubes.
C. The 2 mL suspension contains approximately 1 × 106 cells/mL.
D. Include “Internal” controls – both positive and negative (two of each) with each run. These controls are sperm samples with known DNA fragmentation.
E. The “Kit control” samples, “Test” samples as well as the “Internal” control samples should be centrifuged for seven minutes at 300 × g.
F. Remove 70 percent ethanol with a transfer pipette by aspirating it, without disturbing the cellular pellet.
G. Add 1.0 mL of the “Wash Buffer” (blue cap) and mix well.
H. Centrifuge at 300 × g for seven minutes.
I. Aspirate and remove the supernatant from the tubes.
J. Repeat the washing step with the “Wash Buffer” and discard the supernatant.
K. Number all the tubes starting from the “Negative controls”, “Positive controls”, “Test samples” and “Internal controls”.
19.4.2.4 Staining for TUNEL Assay
A. Count the total number of tubes or test samples including the kit controls and the internal controls.
B. Prepare stain for an additional five to seven tubes.
C. Remove “Reaction buffer” vial (green cap) from 4°C and the TdT (yellow cap) and FITC-dUTP (orange cap) vials (Figure 19.4) from −20°C and place them at room temperature for 20 minutes (Table 19.4). Give a quick vortex to bring the reagent to the bottom of the vial.
D. Prepare the stain as shown in Table 19.5.
E. Add the reagents in the same sequence as indicated in the table.
F. All the steps for the stain preparation must be carried out in the dark.
G. Omit the “TdT” from the “Negative” controls.
H. Resuspend the pellet in each tube in 50 μL of the “Staining solution”.
I. Incubate the sperm suspension for 60 minutesat 37˚C.
J. The tubes should be covered by an aluminum foil.
K. After the 60 minute incubation add 1.0 mL of “Rinse buffer” (red cap) to each tube. Centrifuge the tubes for seven minutes at 300 × g. Aspirate and remove the supernatant.
L. Repeat the wash with addition of 1 mL of “Rinse buffer”.
M. Repeat the centrifugation step for seven minutes at 300 × g.
N. Aspirate and discard the supernatant.
O. Resuspend the pellet in 0.5 mL of PI/RNase buffer.
P. Incubate the suspension mixture for 30 minutes at room temperature.
