The overall goal of the following experiment is to purify at once and at high purity a wide range of mitic cells from mouse adult testes in order to study the dynamic processes taking place during this unique cell cycle. This is achieved by efficiently dissociating testicular cells with a series of enzymatic digestions with collagenase and trypsin in the presence of dna. One to obtain a single cell testy suspension as a second step.
The testy suspension is stained with the Hulk's 3 33 42 fluorescent live DNA dye, which allows various myotic cell types to be distinguished based on their DNA content and density. Next miotic cells are analyzed by fluorescence activated cell sorting in order to identify and purify all the necessary fractions for further analysis such as cytogenetic or gene expression analysis. Results are obtained that show efficient purification of hard to get miotic cell populations at high purity based on comprehensive cytogenetic and expression array studies.
The main advantage of this facts-based protocol over existing methods like centrifugal, literation, or gravity sedimentation, is that it allows to collect at one most miotic fractions at very high purity. Individuals new to this method will have to combine very different skill sets to perform efficient test dissociation and standing followed by optimal cell sorting good interactions between the person preparing the cells and the flow cytometry operator, as well as a good understanding of the fundamental of this protocol essential for success. Demonstrating the cell dissociation protocol will be Dr.I Gatun, a senior postdoc in our laboratory who has considerably improved this protocol.
BVN Tores. Our flow cytometry operator will walk you through an optimal setup for efficient cell sorting. In this protocol, a dissociation of two adult mouse testes will be demonstrated however volumes can be adapted accordingly for juveniles or for additional testes.
To begin at three milliliters of gaze balanced salt solution containing 120 units per milliliter of collagenase type one into the tube Efficient, tasty dis association is crucial for correct stain and trouble resorting of the cells. Addition of sufficient G nase one as well as placement of the tube in a horizontal position during agitation, a very important for efficient test dissociation. Place one encapsulated testis into a 15 milliliter tube.
Add 10 microliters of DNAs one to the testis and shake the tube vigorously by hand until you see the testicular tubule starting to dissociate Next place and secure the tube horizontally on a rotating shaker and agitate the tube at a maximum of 120 RPM for 15 minutes at 33 degrees Celsius after 15 minutes, decant for one minute vertically at room temperature. Then use a disposable pipette to discard the supernatant. Add three milliliters of gaze balanced salt solution containing collagenase type one as well as 10 microliters of D nase one to the sample.
Again, shake the tube a few times by hand. Agitate the tube horizontally at a maximum of 120 RPM for 15 minutes at 33 degrees Celsius as before decant for one minute vertically at room temperature, and then discard the supernatant. Next, add 2.5 milliliters of GBSS containing collagenase.
Type one 50 microliters of trypsin and 10 microliters of dase. One invert the tube several times, agitate horizontally at a maximum of 120 RPM for 15 minutes at 33 degrees Celsius. After the 15 minute agitation, use a disposable plastic past pipette with a wide orifice to gently pipette the sample up and down for three minutes.
No clumps should be visible at this point. Then add 30 microliters of trypsin, 10 microliters of DNAs one and 40 microliters of Hulks 3 33 42 resuspended in DMSO. Invert the tube several times.
Agitate horizontally at a maximum of 120 RPM for 15 minutes at 33 degrees Celsius. When agitation of the sample is complete, add 400 microliters of fetal calf serum and mix by inverting the tube to inactivate the trypsin. To perform the final staining, add 50 microliters of hoax 3 33 42, and 10 microliters of DNAs.
One again, agitate the tube horizontally at a maximum of 120 RPM for 15 minutes at 33 degrees Celsius. The dissociated testis sample is then passed through two 40 micron disposable filters pre wedded with GBSS over a 50 milliliter conical tube. Add five microliters of propidium iodide solution to the sample and mix gently by pipetting several times with a disposable paste pipette.
Next, transfer the sample to a five milliliter plastic syringe through an 18 gauge needle. Then replace the 18 gauge needle with a 22 gauge needle for sample delivery. Finally, store the syringe on ice and protect it from light until the sample is ready for fax processing.
For this experiment, cell sorting is performed on a Backin Dixon area. Two, use cell sorter conditions were adapted from those used previously for a flow cytometer equipped with a 360 nanometer ultraviolet argon laser and a 488 nanometer argonne laser. Ultimately, flow cytometer setup will have to be optimized by cell sorter operator.
Since the Becton Dixon Aria two U does not have a UV laser, the protocol for detecting hawk staining is adapted using a 405 nanometer violet laser. Hooks is excited by the 405 nanometer violet laser and a 488 nanometer blue laser is used for both PI excitation and forward and side scattered detection. In addition, the filters for hos detection are modified in order to limit some red laser leakiness, which results in improved scatter plot sharpness.
The violet laser is configured with a 4 50 40 nanometer band pass for detection of hoaxed blue emission and a 5 85 42 nanometer band pass for hoaxed red emission. A 5 0 2 nanometer long pass is used to separate blue from red fluorescence. The difference from using two different filter sets is shown here.
The 5 85 42 nanometer filter gives more compact cell populations than the 6 30 30 nanometer filter. Therefore, the operator should be ready to try different combinations on their own instrument in order to get the best compromise. A 100 micron nozzle is used with a drop drive frequency of 28, 000 drops per second.
The sample threshold rate is approximately 4, 000 events per second with a flow rate between one to three to maintain greater resolution. Sort the sample in aliquots of approximately 750 microliters dispensed from the syringe During these pauses, keep the remaining sample in the syringe on ice and protected from light. Collect the sorted samples into 12 by 75 millimeter glass borisy located collection tubes containing 250 microliters DMEM supplemented with 10%FCS.
To maintain the sample in collection tubes at four degrees Celsius, use the temperature control option for the entire duration of sorting. Additionally, use the sample agitation feature at 200 RPM to prevent the sample from sedimenting throughout the sort. Each time an aliquot of the main sample is added, it is important to also flick the collection tubes in order to maintain integrity of the collected cells.
Representative facts profiles are shown here where all the major steps of wild type meiosis are identified in this scatter plot where the gated cells are indicated, there's a large amount of debris containing mostly empty membranes and sperm tails present in the adult testes. A representative Propidium iodide plot shows a very limited amount of propidium iodide positive cells present in sample. The typical gait is indicated in this representative wild type hoax 3 33 42 fax profile.
The various myotic cell populations that can be purified using this method for further study are indicated spermatogonia, pre leptin, leptin, zygotes, pacu, diplotene, and round sperate. These last two facts profiles are from a SPO 11 N testis and a wild type testis from 13 day old mice. SPO 11 is the miotic endonuclease that directs doublet strand breaks its sites of miotic recombination hotspots, and as observed here, the absence of SPO O 11 results in a clear miotic failure because no stage past the leptin zygote early PACU can be detected.
In contrast, the profile of a 13 dayed wild type male mouse shows a high concentration of leptin zygote cells. This first wave of myosis is rather asynchronous as clearly visualized. Using this methodology While performing this experiment, it's important to use a sufficient amount of DNAs.
The city's central for trouble free sorting. Although investigators are initially reluctant to use it, the addition of DNA will reduce sample viscosity and enhance cell separation and staining besides intact cells will not be damaged. FOX method for ation of matic cells can be used in a wide range of downstream applications such as, for example, D-N-A-R-N-A extraction to study gene expression dynamics, or for chromatin immunoprecipitation technique to establish various epigenetic profiling.
