Flow Cytometric Analysis
Prior to flow cytometric analysis, the vital dye Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) was added to cell suspensions to a final concentration of 5 μg/mL and incubated for 10 min at 37°C in the dark. Cells were analyzed by means of a MoFlo Cytometer (DakoCytomation) equipped with a UV excitation wavelength laser (Innova 90C-6) operating at 25 mW and a 70-μm nozzle.
Analysis of the following parameters was performed with Summit v4.3 software: forward scatter (FSC-H); side scatter (SSC-H); pulse-area or total emitted fluorescence (FL2-A); and pulse-high or intensity of fluorescence emission (FL2-H). Instrument linearity and doublet discrimination performance was checked with DNA QC particles (Becton Dickinson) stained with Hoechst 33342.
Preparation of Samples for Microscopy
Cell morphology of unstained fresh material was analyzed by phase contrast microscopy with an Olympus IX81 motorized inverted research microscope equipped with a ×40/0.4 UIS2 Plan acromat objective and an Orca AG camera (Hamatsu Photonics C4742–80–12A6). Images were captured using Image ProPlus v.6.0 software.
Results and Discussion
Table 1 shows a comparison between the method presented here for the preparation of testicular cell suspensions and previously described ones [e.g., (7, 9, 14)]. Briefly, the main advantages of our protocol are:
| 1. | Testicular cell suspensions prepared using the Medimachine as described in the “Materials and methods” section are obtained in only 15 min; this includes testis dissection, tissue cutting. and Medimachine processing. This time span represents approximately one-eighth of the time usually taken by other protocols [e.g., (9)]. The brevity of this protocol would account for the good preservation of short-life macromolecules (e.g., some mRNAs), which is critical when a representative sample of compounds present in the original cell population is required. |
| 2. | Since the method involves minimal handling of the material, it is easily reproducible and, again, would prevent RNA degradation during the process. |
| 3. | Unlike most currently used protocols for preparation of cellular suspensions from testis, the method presented here does not involve enzymatic action. The enzymes generally included are collagenase and mainly trypsin which, even though they contribute to an adequate disaggregation of the tissue (3), they can also affect cell integrity and/or preservation of macromolecules of interest. The lack of enzymatic treatments not only favors cell and protein preservation, but also makes the protocol cheaper. |
| 4. | Very little clumping and debris are observed in cellular suspensions. Although it has been previously reported that mechanically prepared cell suspensions clumped more readily than tripsinized ones (3), we have found that our protocol led to well-disaggregated cell suspensions in the absence of enzymatic action (Fig. 1 ). Moreover, the inclusion of NDA (3) all along the process has proved to be very helpful, not only to prevent cell clumping but also to avoid nozzle clogging during flow studies. The good quality of cell suspensions was also evidenced by cytometric analysis, as judged by the minimal cell debris observed for the three examined species (Fig. 2 ). |
| 5. | Very few multinucleates are also observed. According to Meistrich, about 13% of the round spermatids are found as multinucleates either when cell suspensions are prepared by an exclusively mechanical method (14) or in the presence of trypsin (15). Our observations indicate that cell suspensions obtained by the Medimachine method rendered very scarce multinucleates (see Fig. 1 ). This is most probably due to reduced handling, since it has been stated that multinucleates are produced mainly as a consequence of tissue manipulation (3). |
| 6. | The use of a vital dye staining makes the populations suitable for subsequent sorting and RNA extraction. Although Hoechst 33342 concentrations ranging from 1–10 μg/mL and 20–90 min incubation times are usually recommended to obtain DNA histograms with acceptable coefficients of variation (CV), good results were achieved analyzing testicular cell populations with 5 μg/mL dye concentration and reduced incubation time (10 min). The optimal dye concentration and staining time for different cell types vary as dye uptake depends on cellular metabolic rates. Hoechst 33342 noncovalent DNA minor groove binding requires an equilibrium between intracellular free-dye and DNA-bound dye for stoichiometric DNA binding in live cells [(16) and references therein]. Since a short incubation time was desirable to minimize cell death and damage to short-lived macromolecules, this minimal incubation time is considered an important advantage. An additional benefit is the A-T base pair preference of Hoechst 33342 which results in nearly no RNA-associated signal, and therefore no need for RNase treatment. |
Fig. 1 Partial view of a cell suspension from adult rat testis. The suspension was prepared with the Medimachine as described in the “Materials and methods” section and visualized by phase contrast microscopy. The wide variety of cell sizes and shapes can be observed in a well-dissagregated state. As can be seen, most of the spermatozoa keep their flagellae. The absence of multinucleates and the integrity of cell cytoplasms are also evident. The bar corresponds to 25 μm.
Fig. 2 Flow cytometric DNA content analysis of rodent testicular cell suspensions. Cell suspensions were prepared from adult rats (a), mice (b), guinea pigs (c), and from 21-day-old rat pups (d), and stained with the vital dye Hoechst 33342. Three main subpopulations of cells can be easily distinguished in the histograms obtained for adults of the three species (a, b, c), according to their DNA content (C, 2C, and 4C). The additional peak to the left of the C subpopulation corresponds to elongating spermatids and elongated condensed spermatozoa, as previously reported (9). The immature nature of the 21-day-old rat testis (d) is evident, since the C subpopulation is absent.
Table 1 Comparison of different methods for the preparation of testicular cell suspensions
| Lam et al. 1970 | Meistrich 1972 | Malkov et al. 1998 | This work |
|---|---|---|---|---|
Duration | FAST (～30 min) | Time-consuming (～1 h) | Time-consuming (～2 h) | Very fast (～15 min) |
Handling | Significant | High | High | Minimal |
Reproducibility | Variable | Variable | Variable | Very high |
Cell debris | Significant | Moderate | Not shown | Scarce |
Multinucleates | Significant | Significant | Significant | Scarce |
Viability | ≤80% | 98% | Not determined | >85% |
RNAses | No | No | Yes | No |
Trypsin/collagenase | No | Yes | Yes | No |
Even though the Medimachine mechanical protocol proved to be faster and easier than previously described methods, a comparative analysis concerning the quality of cell suspensions still had to be performed. For that purpose, we decided to assess the viability, integrity, and cell type proportions of the cellular suspensions obtained after Medimachine treatment.
The viability of the suspensions was above 85% when tested by means of the Live/Dead kit (Molecular Probes) and the trypan blue dye exclusion test. This was considered totally acceptable, since other mechanical methods used in the past rendered less or, in the best cases, similar viability levels (7, 14). Moreover, the Live/Dead kit’s viability level—which is equivalent to the percentage of intact cells—evidences good cytoplasm preservation (also seen in Fig. 1 ).
On the other hand, the histograms obtained by flow cytometry analysis of testicular cell suspensions from adult rats and mice prepared using the Medimachine mechanical method (Fig. 2 a, b) did not show significant differences with previously described gentle mechanical plus enzymatic protocols (9). This supports the assumption that our method does not selectively damage any specific cell type. Furthermore, when the cellular composition of adult guinea pig testicular cell suspensions obtained through the Medimachine was analyzed by flow cytometry (Fig. 2 c), a coincidence was found with the information we have reported by cell counts in cross sections of seminiferous tubules embedded in Epon (17) (Table 2 ).
Table 2 Comparison of the relative percentages of cell subpopulations in adult guinea pig testes by two methodological approaches
Counts on cross-sections of seminiferous cords (17) | Flow cytometry (this work) |
|---|---|
C?=?66.5% | C?=?65.5% |
2C?=?11.0% | 2C?=?11.5% |
4C?=?22.5% | 4C?=?23.0% |
C, 2C, and 4C refer to DNA content
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