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Method Article
A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.
Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies.
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 .
The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .
1. Preparation of Cell Suspensions
2. Flow Cytometric Analysis
We have used a Becton-Dickinson FACSVantage flow cytometer equipped with a Coherent argon ion laser tuned to emit at 488 nm for the analysis of cells stained with high concentrations of propidium iodide (PI). (The issue of PI entrance into unfixed cells under stress has been addressed elsewhere9,10).
Alternatively, we have employed the vital dye Hoechst 33342 to a final concentration of 5 μg/ml and incubated for 10 min at 37 °C in the dark. Cell analysis was performed by means of a MoFlo Cytometer (DakoCytomation) equipped with a UV excitation wavelength laser operating at 25 mW and using a 70 μm nozzle. Data manipulation was performed with Summit v4.3 software (DakoCytomation).
An example of a well disaggregated cell suspension from rat testes prepared with the protocol described here is shown in Figure 1.
In comparison to enzymatic treatments6,8 and to previously described mechanical disaggregation methods2, the one presented here is much faster, involves less handling, is easily reproducible (not operator-dependant), and renders scarce cell debris (especially compared to other mechanical methods) and very few multinucleates (...
The optimized method described here enables the preparation of cell suspensions from rodent testicular tissue in a very fast and reproducible way, avoiding enzyme and detergent treatment and maintaining good cell integrity and type proportions. The brevity of the procedure (the 15 min span includes testis dissection, tissue cutting, and processing), minimal handling involved, and absence of enzymatic treatments are some of the main advantages. All these would account for the good preservation of short life macromolecules...
This work was partly supported by CSIC (I+D project C022) and PEDECIBA. The authors want to thank Mariela Bollati and Valentina Porro from the Cell Biology Unit (Institut Pasteur de Montevideo) for their generous collaboration concerning the MoFlo cell sorter, and Merial-Montevideo for gently providing all the guinea pig specimens used in this project. Figure 1 was reproduced with permission of BioMed Central; Figures 2 and 3, and Table 1 with permission of S. Karger AG, Basel; and Figures 4 and 5 were reproduced or adapted with permission of John Wiley & Sons, Inc (copyright owned by Wiley-Blackwell, 2011).
Name | Company | Catalog Number | Comments |
Name of Reagent/Material | Company | Catalogue Number | Comments |
Medimachine system | BD | 340587 | |
Medicon unit (50 μm) | BD | 340591 | |
Filcon unit (50 μm) | BD | 340603 | |
DMEM | Gibco | 430-2100 | |
Fetal calf serum | PAA | A11-151 | |
NDA | Chemos BmbH | 277081 | |
Hoechst 33342 | Sigma-Aldrich | 14533 | Stock solution 5 mg/ml; used at a final concentration of 5 μg/ml |
Propidium iodide | Sigma-Aldrich | 287075 | Stock solution 1 mg/ml; used at a final concentration of 50 μg/ml |
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