In this video, a very simple and efficient protocol to obtain cellular suspension of test material from rodents in only 15 minutes will be presented. The method has been applied to test test from mouse, rat Guinea pigs and uses a meta machine device to disaggregate the encapsulated tissue. After a filtration and staining the cell inion is ready for flow.
Cyto analysis or sorting Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male GA differentiation. Thus, testicular heterogeneity with over 30 different cell types represents one of the major problems concerning the study of the molecular basis of mammalian spermatogenesis. For that reason, various methods have been used to obtain enrich to purified testicular cell populations, including PU and centrifugal illustration.
The first step for any cell separation method is the preparation of a cellular suspension. However, current protocols are usually time consuming and may imply loss of short-lived molecules such as RNAs, which is undesirable for ulterior expression analysis. The main advantage of the protocol presented here is that eliminates steps between animal sacrifice and sperma stage specific molecular studies, but at the same time ensures good quality and excellent cell type representation in the cellular suspensions.
Demonstrating the procedure will be Dr.Ana Rodriguez, researcher at AL Laboratory who developed the method and Federico San Lopez flow cytometry that were in charge of the cytometric analysis. The procedure requires a small piece of equipment, the meach system, with its medicon and fcon accessories. However, the fcon accessories can be perfectly replaced by nylon membranes of the same pore size.
The only other required materials and reagents are glass spread, two dishes, scissors and forceps, A three to five milliliter syringe annoy bowel chamber, DMM culture medium supplemented with 10%fetal cough serum and NDA. If downstream applications involving RNA manipulations are planned, then all the materials and solutions should be treated accordingly to perform the protocol. Place the dissected testis in a 96 millimeter glass per data shown ice containing 10 milliliters of supplemented ice called dm.
Remove the TAL Virginia, cut the decap testis into square pieces of two to three millimeters on each side. Place four to five of these pieces in a disposable desegregated medicon along with one milliliter of code supplemented D and processed in the meta machine system for 50 seconds. Each medical unit contains a fixed stainless steel screen with about a hundred hexagonal holes surrounded by six micro blades.
The tissue is brought to each hole by a metal rotor inside the medicon chamber and desegregated by passing over the sharpened holes and through the metal screen while a micro pump under the screen supplies liquid and flushes out the hulls. Recover the result in cell suspension from the medicon using a three to five millimeter syringe without needle. Subsequently soak a 50 micrometer falcon or same four size nylon mesh with half a milliliter medium and filter the suspension through it.
Repeat the step, but using a 25 micrometer nylon mesh or equivalent falcon unit and place on ice, take an ALI quote of the cell suspension to count in a noyer chamber and adjust cellular concentration to one to two times 10 to the seven cells per milliliter in culture media. Finally, add NDA to a final concentration of 0.2%to avoid cell pumping and also to avoid muscle clogging during subsequent flow studies. If cell viability is intended to be evaluated, this can be performed by means of the live dead viability kit for animal cells following the instructions of the manufacturer prior to flow cytometric analysis, add a fluorescent dye.
The fluorochrome choice will depend on available lasers. In this demonstration, the use of different dyes that binds document to DNA will be shown on. Although propidium yo diet is the usual dye for fixed or dead cells at low concentrations, we have found that when used at high concentrations, the ROM enters unfix testicular cells in the suspensions prepared by the method presented here.
This is probably due to the mechanical stress implied, which in combination with the sensorial nature of the semial epithelium could lead to breakage of cell to cell connecting bridges with minimal manipulation For propidium iodide staining 10 minutes prior to sample analysis at the solution at a final concentration of 50 micrograms per milliliter to the cell suspension and incubate a zero degree Celsius in the dark For analyzing propidium mu stain cells. For further sorting, we use a fax vantage flow cytometer equipped with a coherent targon ion laser tuned to emit at 488 nanometers. Laser power is set to a hundred milliwatt and the propidium I emitted fluorescence is collected an FL two detector using a 5 7 5 slash 26 band pass filter.
Use a 70 micrometer nozzle for flow cytometry measurements and squa software to analyze the following parameters. Forward scatter side scatter, total emitted fluorescence or pulse area and duration of fluorescent emissions or pulse width. Results are presented in grams representing the number of events at each fluorescent level and in D plots displaying side scatter versus fluorescent pulse area and fluorescent pulse area versus its width.
Doublets are excluded from the analysis using DO plots of fluorescent pulse area versus its width for sorting SM monocyte populations with the fax vantage set sorting mode in normal R or normal C and use three sorted drops as envelope. Adjust sample differential to analyze cells at a rate of 500 to 1500 per second. Keep sample and collection tubes three to four degrees celsius by using a refrigeration unit.
Alternatively, cells in the suspension can be stained with other fluorochromes. For instance, the vital dye. HER 3 3 3 4 2 can be used to a final concentration of five micrograms per milliliter and incubated for at least 10 minutes at 37 degrees protected from light for Hirsch.
3, 3, 3, 4, 2 stain samples. Cell analysis has been performed by means of a MO flow cytometer equipped with a UV excitation wavelength laser operating at 25 milliwatts and a 70 micrometer nozzle. Use Summit 4.3 software or similar to analyze the same parameters indicated before when the cell suspensions are analyzed under face contrast optics cut quality suspensions are observed.
The main advantages of this protocol are in the first place testicular cell suspensions prepared using the many machine and obtaining only 15 minutes including test C-section, tissue cutting, ach, machine processing and filtration this time span about an eighth of the time usually taken by other protocols. The brevity of this protocol together with a minimal handing involved would account for the good preservation of short life macromolecules, which is critical when a representative sample of compounds present in the original cell population is required. As the methane involves minimal handling, it is easily reproducible in this table.
The relative percentages of the different testicular cell populations for seven independent experiments performed with adult rats and analyzed by flow cytometry are presented as an example to show reproducibility of the method. Unlike most currently used particles for preparation of testicular cell suspensions, the method presented here does not involve enzymatic action. Although collagenase drip in an RNAs, which are the generally included enzymes contribute to inadequate to segregation of the tissue, they can affect the preservation of macromolecules of interest.
The lack of enzymatic treatments not only favors RNA and protein preservation, but also makes a protocol cheaper. Though it had been previously reported that mechanically prepared cell suspensions clumped more readily than tryps inized ones, we have found that this protocol leads to well desegregated cell suspensions in the absence of enzymatic action me. The inclusion of NDA has proven to be very helpful in preventing cell clumping.
The good quality of the suspensions is also evidenced when the samples are subjected to cytometric analysis as judged by the minimal cell debris observed. According to published information, about 13%of round sperms are found as multinucleate either when cell suspensions are prepared by an exclusively mechanical method or in the presence of trypsin. However many machine prepared suspensions render very scarce multinucleate most probably due to reduce tangling since it has been stated that multinucleate are produced mainly as a consequence of tissue manipulation.
An example of a many machine prepared cell suspension from rat testis is observed in this picture. As can be seen. Cytoplasms are well preserved the relative percentages of C two C and four C cell populations in many machine prepared testicular suspensions for different rodents reassembled those of the intact PEs.
This supports the assumption that the method described here does not selectively damage any specific cell type. Surprisingly cell type representation in many machine prepared suspensions is much better maintained than with the classic drippin preparation methods. As can be seen, cyto graphics obtained for meine processed testicular cell suspensions do not substantially defer from previously reported ones.
In fact, the obtained histograms and dot plots exhibit the three characteristic four C to C and C testicular cell populations based on DNA content as well as the apparently subha population, which represents the highly condensed haploid spermatozoa. These profiles were observed either for her sustained or propidium iodide stain suspensions respectively, and this stands for adult and for juvenile specimens as well. Sorting of sper mitogenic cells has been successfully achieved with over 98%purity, either for selected testicular populations based on DNA content or even for subpopulations with the same DNA content, but different chromatin condensation level such as sleep tine and tine myocytes.
As we could observe in Guinea pig test suspensions, as the protocol does not include RNA treatment sorted populations render good RNA quality and allow differential gene expression studies even for propidium iodide stained populations. In that sense, it is worth reminding that unlike other dyes, propidium iodide stains, DNA as well as double stranded RNA, however, in this graphics of a Guinea pig testicular suspension analysis, the similar profiles obtained using propidium iodide in parallel with vibrant ized cycle orange. That is a vital DNA specific dye show that RNA staining does not significantly influence cytometric histogram patterns.
This list summarizes the main advantages of the percented protocol. The optimized method described here allows to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different sperm stages from rodents in only 15 minutes, including testis dissection, tissue cutting and processing through the ME machine system. The brevity, higher reproducibility quality and cell type proportions in the cell suspensions make it a good alternative for other, more tedious and time consuming self preparation techniques currently in use.
Moreover, since this method involves very little manipulation and avoids enzymes and detergents, it is an ideal choice for delicate downstream application such as gene expression studies.
