The overall goal of this procedure is to separate mouse spermatids into four distinct populations, allowing the molecular study of spermiogenesis. This method can, can help answer key questions in the mirror reproduction field, such as what is the impact of chromatic modeling on genomic integrity. The main advantage of this technique is that it provides separation of sperm into pure cells population without cross-contamination.
We first had the idea for this method when we observed important variations in the DN staining intensity during chromatin remodeling in haplo parameters On the day before the cell sorting. Add one to two milliliters of heat inactivated FBS to five milliliter polypropylene round bottom, and 15 and 50 milliliter polypropylene conical tubes. Next slowly coat the tubes by overnight inversion at four degrees Celsius on a rotator the next day.
Carefully decant the FBS using a P 200 pipette to remove any residual FBS from the 15 and 50 milliliter tubes and leaving a small residual volume of 100 to 200 microliters of FBS in the five milliliter tubes for collecting the purified cells on the day of the cell. Sorting, mince the encapsulated test pieces in 500 microliters of sorting buffer. Then transfer the tissue fragments into a 1.5 milliliter tube using a truncated micro pipette tip.
Flush the germ cells from the seminiferous tubules with gentle up and down pipetting, followed by flushing with an intact tip. Next, use a 40 micron cell strainer to remove the debris and clumps collecting the filtrate in an FBS coated 50 milliliter conical tube. Wash the filter once with sorting buffer up to a total volume of three milliliters and transfer the filtrate into an FBS coated 15 milliliter conical tube.
Then add 16 microliters of EDTA per milliliter of cell suspension to the cells and use a 10 milliliter pipette to slowly fix the cells over one minute with three volumes of ice cold, 100%ethanol while low speed vortexing, the suspension will become milky after fixation. Incubate the cells on ice for 15 minutes with occasional inversion. Then centrifuge the cell suspension, resuspend the pallet in two milliliters of sorting buffer and stain the germ cells with four microliters of cyto 16 DNAD immediately before sorting.
Filter the cells through a 50 micron filter into an FBS coated fax tube and wash the filter extensively with one to two milliliters of sorting buffer. Load the fixed germ cells onto a 488 nanometer laser equipped cell sorter. Next, display the total events with a histogram representing the number of events against the Alexa floor 4 88 area and gate the positive DNA staining cells.
Then display the cells from this gate in a side scatter area against a forward scatter area plot and gate. The cells as indicated, display these gated cells in a forward scatter area against an Alexa floor 4 88 area, plot and gate. The cells again then display these gates into separate Alexa floor 4 88 width against Alexa floor 4 88 area plots.
The spermiogenesis steps one through nine and 10 through 12. Spermatids can then be gated for sorting to collect steps 13 through 14 and 15 through 16. Spermatids display the cells from the first gate into a forward scatter area against an EXOR 4 88 area plot and gate.
The cell populations as indicated. Then display these gates into individual Alexa. Floor 4 88 width against Alexa floor 4 88 area plots and gate.
The steps 13 through 14 and 15 through 16 sperms for sorting. Finally collect the purified fractions in 100 to 200 microliters of FBS in individual FBS coated five milliliter round bottom tubes on ice. In these images dappy staining of the flow sorted smited populations is shown.
The spermiogenesis steps one through nine spermatids typically display round shaped nuclei that appear oval shaped in spermiogenesis. Step nine spermatids, and that are observed as elongated hooks in steps 10 through 12. Spermatids spermiogenesis steps 13 through 14 and 15 through 16 spermatids exhibit nuclei with a similar shape, but with a slightly differing DNA staining intensity, which allows these distinct sper populations to be identified during their sorting by flow cytometry.
Following the demonstrated protocol, the purity of the individual S experimented populations can be expected to be between 95 and 100%This technique can be completed in six to eight hours. If each step is followed properly. It is to remember to always keep the cells on ice throughout the procedure by following this procedure for distinct populations of sperms can be obtained, providing a sufficient amount of material for molecular analysis, such as characterizing the dramatic ting remodeling process or to study the genetic impact of this transition.
