Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

This new efficient method for isolating subpopulations of spermatocytes and spermatids from adult mice can be used to investigate the molecular mechanisms underlying meiosis and spermatogenesis. This method uses a low cytotoxicity DNA binding dye with a wide excitation spectrum that can be applied to most currently used FACS orders. This method is very straightforward.

The most challenging aspect is the flow cytometry gating but the detailed gating strategy should help with adapting the protocol to other cells sorters. Demonstrating the procedure will be Yu-Han Yeh, a research assistant from Satoshi Namekawa's Laboratory. After harvesting both testes from an eight week old male mouse, place the tissues in a 60 millimeter Petri dish containing two milliliters of ice cold PBS and remove the tunica albuginea.

Gently separate the testes with forceps to slightly disperse the seminiferous tubules. And transfer the tubules into a drop of fresh ice cold PBS in a 100 millimeter Petri dish. Use the forceps to gently untangle the tubules and wash the tubules in three additional droplets of PBS as just demonstrated.

After the last wash, place the untangled seminiferous tubules into a 15 milliliter tube, containing two milliliters of dissociation buffer for a 20-minute incubation at 37 degrees Celsius. At the end of the incubation, use a 1000 microliter pipette to gently pipette the tubules 20 times, before returning the tube to the incubator for an additional six minutes. At the end of the incubation, pipette the tissues an additional 20 times, before returning the tube to the incubator.

After three minutes, gently pipette the tubules 10 times or until no visible tissue pieces remain, before stopping the dissociation with 10 milliliters of FACS buffer. Then, centrifuge to the tissue suspension two times using an additional 10 milliliters for fresh FACS buffer for the second wash to remove the spermatozoa and as much debris as possible. To stain the cells for flow cytometric analysis, re-suspend the pellet in three milliliters of FACS buffer.

And filter the cell suspension through a 70 micron nylon cell strainer, into a 50 milliliter tube. After counting, transfer 10%of the cells into a new tube on ice as the unstained negative control. And thoroughly mix six microliters of DCV stain into the remaining cell suspension.

After a 30-minute incubation at 37 degrees Celsius in the dark with gentle shaking every 10 minutes, add five microliters of DNase I to the cells. And filter the cells through a 35 micron nylon mesh strainer into a five milliliter FACS tube on ice. For flow cytometric analysis of the cells, create a new experiment in the flow analysis software, and under the worksheet tools menu, click new density to create a forward scatter area versus backscatter area density plot on a linear scale.

Click new histogram to create a DCV blue histogram plot on a logarithmic scale. And click start and record to begin processing the unstained sample. While the sample is running, click detector and threshold settings to adjust both the forward and side scatter PMT voltages to place the unstained cells on the scale of the forward and back scatter plot.

Adjust the PMT voltage for the jappy channel to locate the position of the DCV negative population in the first decade of the DCV blue histogram logarithmic plot. Then, click stop to unload the unstained sample. After briefly vortexing and loading the stained sample, click next tube to create a new worksheet for the sample.

Set the cytometer to acquire at least one times 10 to the six events, and click start and record. Next, click new density to create forward scatter height versus forward scatter width. And DCV blue versus DCV red density plots on a linear scale.

On the forward scatter area versus backscatter area density plot, use the polygon tool to draw a gate called cells to include most of the cells and to exclude small debris. Apply this gate to the forward scatter height versus forward scatter width plot and use the rectangle tool to draw a single cell's gate to exclude non single cells. Apply the single cell's gate to the DCV blue versus DCV red density plot and adjust the scale to capture an extended profile.

Use the polygon tool to draw a DCV gate to exclude the unstained cells and side population, and apply the gate to a DCV blue histogram plot on a linear scale. The three major peaks refer to the different 1C, 2C and 4C DNA contents. Create a second DCV blue versus DCV red density plot on a linear scale.

And back gate the DCV gate onto the plot to locate the 1C and 4C populations. Create another DCV blue versus DCV red density plot on a linear scale. And use the ellipse tool to draw a gate on the 1C population.

Use the polygon tool to gate the 4C population with a continuous curve. And create another DCV blue versus DCV red density plot on a linear scale. Use the polygon tool to create a 4C one gate.

In a new forward by side scatter plot, use the ellipse tool to create pachytene, diplotene and leptotene, zygotene spermatocyte gates. When all of the gates have been drawn, create a new DCV blue versus DCV red color dot plot on a linear scale to apply the for 4C gate to ensure that the three populations are in a continuous order within the gate. Next, create a new forward scatter area versus backscatter area density plot on a linear scale, and select the unified size of cells as a pure round spermatid population.

After sorting, the representative purities of the separated leptotene, zygotene, pachytene and diplotene spermatocyte fractions are typically between 80%to 90%as confirmed by SYCP3 and Gamma H2AX immunostaining. The purity of the round spermatid faction can be confirmed by nucleus staining with DCV in combination with Sp56 and H1t staining, and is also typically around 90%The viability of the isolated cell populations is usually over 95%And the average total yields of each fraction from a single adult mouse is typically sufficient for various downstream analysis. The sorted cells can be used for various next generation sequencing analysis to better explore gene expression and regulation during spermatogenesis.