Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

The overall goal of this procedure is to characterize and quantify the number of peripheral blood mononuclear cells that have infiltrated the early brain tumor microenvironment. This method can help address key questions in the field of brain tumor immunology by identifying factors important for the trafficking of peripheral immune cells into the brain tumor microenvironment. The main advantage of this technique is that it yields time dependent quantitative data on the number and activation status of leukocytes that have entered this CNS.

After placing an anesthetized mouse into a stereotactic frame, make a two centimeter midline incision over the cranium from the frontal bone to the occipital bone using a scalpel blade. Then retract the skin at the incision using Collibra retractors. After that, lower a microliter syringe equipped with a 33 gauge needle to directly above the bma.

Adjust the position of the needle to reach the target site for tumor implantation. Then use a one milliliter tuberculin syringe equipped with a 26 gauge needle to mark this location by gently excoriating the periosteum after that. Drill a hole into the cranium at the target site with the 0.8 millimeter drill bit until it reaches the underlying dura mater while drilling.

Apply intermittent pressure followed by quick removal of the drill bit from the cranium surface to avoid excessive heat and force. Next, flush the microliter syringe with 0.9%sodium chloride in a 1.7 milliliter conical polypropylene micro tube to ensure that the needle is not clogged. Gently flick the 0.6 milliliter conical polypropylene micro tube containing the tumor cells several times to thoroughly resus suspend the cells.

Then draw up two microliters of cells into the microliter syringe and ensure no air bubbles are introduced into the syringe. Dispense one microliter of the cells onto a 70%isopropyl alcohol prep pad, leaving exactly one microliter of tumor cells in the syringe. After afterward, bring the needle down until it touches the dura mater.

Then lower the needle 3.5 millimeters into the brain, and subsequently retract the needle by 0.5 millimeters slowly and smoothly. Deliver the cells by depressing the syringe plunger over the course of one to two minutes. To perform trans cardial perfusion on the deeply anesthetized mouse.

Insert a blunt needle into the left ventricle of the heart. Use a small pair of dissection scissors to snip the right atrium to allow for exsanguination perfuse, the mouse circulatory system with tyro solution for several minutes until the liver and lungs have completely blanched to the lack of blood. Ensure that no gross amount of blood continues to exit the right atrium.

Prior to removing the 20 gauge aluminum hub blunt needle from the left ventricle. After removing the needle, unpin the mouse from the extruded polystyrene foam block and turn the mouse ventral side down. Using a large pair of dissection scissors, separate the head from the rest of the body.

Then cut the scalp on the midline using a small pair of dissection scissors working from the occipital bone to the snout in order to expose the cranium. Subsequently retract the skin on both sides of the cranium and hold the skull securely. Use a pair of bone URS to carefully break through the cranium beginning at the occipital bone and work forward to completely expose the dorsal and lateral surfaces of the brain.

Harvesting of the mouse brain from the cranium without causing damage to it may initially prove difficult. Those inexperience with the technique may wish to try several times prior to running proper experiments. Once the cranial bones have been removed, turn the head of the mouse ventral side up.

Cut the cranial nerves at the base of the brain in order to free it from the skull. In this procedure, using a clean single edged razor blade isolate the area of the brain containing the tumor by making a sagittal cut down the center of the brain. To dissect the two hemispheres, turn the ipsilateral hemisphere medial side down and make one coronal cut at the cerebellum and another cut at the olfactory bulb to isolate the target tissue containing the tumor implant.

Next, place the target tissue into the labeled glass down tissue grinder containing one milliliter of DPBS. Push the plunger all the way down and twist seven times to disrupt the tissue. After that, lift the plunger to allow the liquid to settle back to the bottom of the tissue grinder.

Repeat this step thrice. However, only twist the plunger four times during the next two repeats and three times during the last repeat. To avoid over tating the brain tissue.

Subsequently apply three one milliliter volumes of ice cold DPBS to the sides of the plunger to rinse the residual brain matter into the tissue grinder. Then we suspend the TRID brain matter by pipetting up and down, and place into a labeled 15 milliliter centrifuge tube on ice. Rinse the sides of the down tissue grinder with an additional one milliliter of ice cold DPBS and add to the same 15 milliliter centrifuge tube.

Keep all the tubes containing TRID brain matter on ice until all the samples have been processed. Then spin down the TRID brain matter in the 15 milliliter centrifuge tubes at 740 times G for 20 minutes at four degrees Celsius. Next, remove the supernatant and re suspend the pelleted brain matter in one milliliter of a previously prepared mixture of collagenase and DNA one digestive enzymes.

Place the 15 milliliter centrifuge tubes into a test tube rack and place the rack in a 37 degree cell CS water bath for 15 minutes. Then gently agitate the samples twice throughout the incubation period by flicking the tubes to facilitate tissue disaggregation. Subsequently, add six milliliters of vice cold DPBS to each tube to dilute the digestive enzymes.

Pipe that up and down to resuspend and filter the total volume through sterile 70 micron nylon mesh filters into new labeled 15 milliliter centrifuge tubes on ice. Afterward, spin the brain cell suspension down at 740 times G for 20 minutes at four degrees Celsius to achieve a single cell pellet. Remove the 15 milliliter tubes from the centrifuge and place them on ice.

Then increase the temperature setting on the centrifuge to about 21 degrees Celsius in preparation for the next step. Next, fully remove the cell snat and initially resuspend the cell pellets in one milliliter of 70%density centrifugation media using a P 1000 micro pipette. Then add four additional milliliters of 70%density centrifugation media to each tube.

Put the caps on securely and homogenize the cell suspension by gently inverting the tube several times after that transfer the 15 milliliter centrifuge tubes containing brain cells resuspended in 70%density. Centrifugation media from ice to a test tube rack at room temperature one at a time. Carefully overlay two milliliters of 37%density centrifugation media solution onto the five milliliters of 70%density centrifugation media to form a clean interface between the two density centrifugation media layers.

Then mark the position of the interface between the two layers with a fine tipped marker so that it can be easily identified after centrifugation when the distinction becomes less apparent. Spin down the 15 milliliter centrifuge tubes at 740 times G for 20 minutes at room temperature with no break. To avoid disrupting the interface between the density centrifugation media layers afterward, collect the pbmc that have accumulated at the interface between the two density centrifugation media layers by introducing a P 200 micro pipette into the tube.

Carefully bypassing the lipid layer once at the level of the PBMC band, slowly extract 200 microliters from the surface of the 70%density centrifugation media layer and transfer to a respectively labeled polypropylene fax tube on ice. Repeat this step once to achieve a total volume of 400 microliters per sample. The experimentalist ability to form a clean interface between the two density centrifugation media layers is crucial to the reproducible isolation of p BMCs.

One should pour the 37%density centrifugation media slowly while angling the tube 20 to 30 degrees above the bench top. This figure shows the gating strategy for a typical experiment gates placed on GR one positive CD 11 B positive myeloid cells shown in step five and NK 1.1. Positive natural killer cells shown in step six are then stratified based on GZMB expression in steps five prime and six prime respectively.

In this figure back gating GR one positive CD 11 B positive myeloid cells and NK 1.1 positive natural killer cells onto FSCA versus SSCA demonstrates the smaller lymphoid size of natural killer cells compared to the relatively larger sized myeloid population, thus providing further confirmation of the identity of these two pbmc subpopulations. The data here shows that engraftment of GL 26 sital one I glioma cells into the syngen. A XC 57 black six J mouse brain rapidly induces the recruitment of CD 45 positive PBMC numbers shown in association with each data point along the GL 26 sit GAL one I curve represent the fold induction of tumor infiltrating pbmc above control.

GGL 26 sit NT tumors at the specified hour post tumor implantation or HPI based on the specific antibody cocktail used in the demonstration GR one positive CD 11 B positive myeloid cells and NK 1.1 Positive natural killer cells were shown to specifically enter the GAL one deficient tumor microenvironment within 48 hours of tumor engraftment. However, the total number of glioma infiltrating myeloid cells far outweighed that of natural killer cells. Once mastered, the methodology is outlined from brain harvesting to flow.

Cytometric analysis can be performed in roughly six hours by one experimentalist. It is imperative that the entire volume of each PBMC sample be analyzed by the flow cytometer in order to achieve a fair comparison of the total number of glioma infiltrating pbmc complementary experiments. Such immunohistochemical analysis may be performed to obtain spatial data on the immune cell infiltrates and to allow for measurement of overall tumor size and microanatomy at time points that correspond to those assessed using this procedure.

This technique may be applied to the study of diverse brain tumor models, including those generated de novo with the use of oncogene encoding plasma DNA without significant alterations to the overall procedure. After watching this video, you should have a good understanding of how to prepare glioma cell suspensions for stereotactic and graftman into a mouse brain, how to transi profuse tumor, brain bearing mice, and how to isolate and flow cyto metrically, analyze glioma infiltrating peripheral blood mononuclear cells.