Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

This method can help answer key questions in lymphatic and liver fields, such as how lymphatic endothelial cells, or LECs, respond to specific stimuli and how that contributes to disease pathogenesis. The main advantage of this technique is that we can evaluate liver lymphatic endothelial cell phenotype and function on a per cell basis, and can be used for down-stream applications after cell sorting. Though this method can provide insight into liver lymphatic biology, it can also be used to evaluate lymphatic endothelial cells from different tissues, such as skin and mammary gland.

Demonstrating the procedure will be Jeffrey Finlon, a professional research assistant from my laboratory. To prepare a single-cell suspension from isolated liver cells, begin by spraying down the mouse with seventy-percent ethanol, and securing the paws to a dissection board. Use dissection scissors to cut the skin about one centimeter above the anus and use toothed forceps to lift the skin away from the body.

Insert the scissor tips between the skin and the peritoneum and open the scissors to separate the tissues before continuing the skin incision up to the neck. Secure the skin to the dissection board under each forelimb and above each hindlimb, and pull the peritoneal sack upward to extend the incision toward the neck. Grasp the lobes of the liver and cut just below the sternum to allow dissection around the liver.

Then transfer the organ into four milliliters of Click's EHAA medium. Use the scalpel to cut the liver into approximately one-millimeter diameter pieces and digest the pieces in five hundred microliters of freshly prepared digestion solution and five hundred microliters of DNase 1. After fifteen minutes at thirty-seven degrees Celsius, use a five hundred milliliter pipet to mix the tissues thoroughly and return the container to the incubator for another fifteen minutes.

At the end of the incubation, transfer the digested sample through a one hundred micron strainer into a fifty milliliter conical tube and use the plunger from a one milliliter syringe to gently press the remaining pieces of tissue through the mesh. Wash the filter with five milliliters of isolation buffer and gently press any remaining tissue through the strainer. Collect the isolated liver cells by centrifugation and re-suspend the pellet in four milliliters of red blood cell lysis buffer.

After five minutes at room temperature, wash the cells in ten milliliters of isolation buffer and count the cells on the hemocytometer to determine the full liver count. Re-suspend the pellet in five milliliters of twenty-percent iodixanol and overlay the cell suspension with one milliliter of PBS. Separate the cells by density gradient separation and harvest the layer between the PBS and the iodixanol.

Then filter the cells through a one hundred micrometer strainer into a new fifty milliliter conical tube. Wash the cells in ten milliliters of fresh isolation buffer and re-suspend the resulting pellet in five hundred microliters of PBS, supplemented with two-percent fetal bovine serum, or FBS. After counting, aliquot approximately five times ten to the sixth non-parenchymal cells into each control and experimental well of a ninety-six well plate for centrifugation and re-suspend the pellets in ninety microliters of PBS plus FBS per well.

Add the appropriate experimental and control primary antibody cocktails to each well and stain all of the wells with an appropriate viability marker. The most important step in this procedure is the proper titration of the antibodies and the use of ice type controls and fluorescence-1 controls to validate the staining. After thirty minutes at four degrees Celsius, centrifuge wash the cells with one hundred microliters of PBS plus FBS per well and transfer the cells from each well into individual five milliliter round-bottom flow cytometry tubes to a four hundred microliter final volume of PBS plus FBS per tube.

Then, use a small volume of cells to adjust the laser and compensation settings on the flow cytometer and read all of the events for each tube. When all of the tubes have been read, import the data into an appropriate flow cytometry analysis program. Gate on live cells based on the viability marker dye where negative expression is considered live.

Gate on the live cells based on the size and granularity of the cells as well as their viability marker dye expression, followed by gating on the CD45 negative CD31 positive cell population using the appropriate isotype control and fluorescence-1 data to determine the positive and negative populations. Then gate the CD45 negative CD31 positive cells according to their CD16/32 and podoplanin expression to allow identification of the CD45 negative CD31 positive, CD16/32 negative podoplanin positive lymphatic endothelial, and CD45 negative CD31 positive, CD16/32 positive podoplanin negative liver sinusoidal endothelial cell populations. Emphatic endothelial cells express lymphatic vessel and endothelial hyaluronan one, but not CD146.

Liver isolated CD31 positive CD16/32 positive cells isolated as demonstrated are also CD146 positive and podoplanin positive, while the CD31 positive CD16/32 negative cells are primarily CD146 negative and either podoplanin positive or negative. Positivity was determined based on fluorescence-1 staining. The liver lymphatic endothelial cells are CD146 negative and PDPN positive.

Neither the vascular endothelium nor murine liver macrophages express podoplanin. Podoplanin staining can also be used to distinguish cholangiocytes from lymphatic vessels based on the distinct nuclear structures of the bile ducts as well as by cytokeratin 7 staining. As only lymphatic endothelial cells co-express vascular endothelial growth factor receptor 3 and Prospero homeobox protein 1 in the liver, these markers can be further used to confirm the purity of the sorted liver lymphatic endothelial cell population.

While attempting this procedure, it is important to remember to work quickly and keep cells on ice. Following this procedure, other methods like flow sorting of LEC populations can be performed in order to answer additional questions, such as transcriptional profiling of LECs. After its development, this technique paved the way for researchers in the field of liver-specific lymphatic biology to explore how lymphatics impact liver disease in mice and humans.

Generally, individuals new to this method will struggle because lymphatic endothelial cells are an infrequent, gradual population in a liver and the method needs to be performed quickly.