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

Summary

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Abstract

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the β-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1⁺/CD11b⁺ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists’ discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Introduction

Gliomas are a class of neuroepithelial brain cancers arising from transformed glia within the central nervous system (CNS). Of all the gliomas, World Health Organization (WHO) grade IV glioma, or glioblastoma (GBM), is the most common and lethal¹. GBM is highly refractory to the current standard-of-care which consists of tumor resection to the extent possible followed by radiation plus concomitant and adjuvant chemotherapy with temozolomide². These deadly cancers carry a dismal prognosis of only 15-18 months of survival from the time of initial diagnosis with only 5% of patients surviving the disease after 5 years³.

The presence of the blood brain barrier (BBB), lack of professional antigen presenting cells (APCs), and the previously unidentified existence of bona fide lymphatic structures within the brain⁴ have led to the notion of GBM as immune privileged. However, numerous studies now show that these brain cancers indeed engender the recruitment of peripheral immune cells that are predominantly myeloid in origin which include monocytes, macrophages, and myeloid-derived suppressor cells (MDSCs)⁵. GBM also influences the activity of brain-resident microglia to become pro-tumorigenic^6,7. Lymphoid cells such as CD8⁺ T cells⁸ and CD56⁺ natural killer cells⁹ are also present within the tumor microenvironment, but in much fewer numbers, a fact thought to be due to immunosuppressive function instigated by glioma-derived factors on tumor associated macrophages (TAMs)¹⁰. CD4⁺ T cells are also present in GBM, but much of this population also expresses CD25 and FoxP3, makers of immunosuppressive T regulatory (T_reg) cells¹¹. The overall immunosuppressive state of GBM culminates in the promotion of immunologic escape and tumor progression¹².

A better understanding of the mechanisms of GBM immunosuppression is critical to the development of effective immunotherapeutic strategies designed to stimulate the immune system against the tumor. Over the last 15 years our lab has worked to overcome the mechanisms of brain tumor immunosuppresson in order to develop efficacious new anti-GBM immunotherapeutics^13-19. The culmination of this work has now led to a clinical trial designed to evaluate a combined cytotoxic and immune-stimulatory therapeutic for patients with newly diagnosed GBM (ClinicalTrials.gov Identifier: NCT01811992).

Our most recent work shows that mouse GL26 and rat CNS-1 GBM cells block anti-tumor NK cell immune surveillance by producing large amounts of the β-galactoside-binding lectin galectin-1 (gal-1)²⁰. This was demonstrated by suppressing the expression of gal-1 in glioma cells using shRNA-mediated gene knockdown. In vitro experiments showed that gal-1-deficient glioma cells proliferated normally in culture, yet underwent rapid rejection soon after intracranial engraftment into syngeneic C57BL/6J or RAG1^-/- mice, thus establishing the independence of T- or B- cells on this form of tumor rejection. NK cell immunodepletion with anti-asialo GM₁ anti-serum or monoclonal NK1.1 antibodies led to the complete restoration of intracranial gal-1-deficient glioma growth, establishing the role of NK cells in gal-1-deficient glioma rejection. We now show that immunodepletion of Gr-1⁺/CD11b⁺ myeloid cells is sufficient to prevent gal-1-deficient glioma rejection despite the presence of NK cells, thus revealing a indispensible auxiliary role for myeloid cells in the aiding of NK-mediated gal-1-deficient tumor lysis (unpublished data). This unexpected result has led us to develop a comprehensive protocol for the isolation and analysis of peripheral blood mononuclear cells (PBMCs) that infiltrate the brain tumor microenvironment soon after intracranial engraftment so that we may better characterize the immune infiltration events that predicate gal-1-deficient glioma rejection.

The method is demonstrated here by using mouse GL26 glioma cells that constitutively express mCitrine fluorescent protein, called GL26-Cit, which permit direct tumor cell visualization by fluorescence microscopy²¹. These cells are stereotactically engrafted into the brain of syngeneic C57BL/6J mice and are allowed to grow for 24, 48, or 72 hr prior to mouse euthanasia. Glioma-infiltrating PBMCs are then isolated and immunolabeled using anti -CD45, -Gr-1, -CD11b and -NK1.1 cell surface antibodies together with intracellular immunolabeling for granzyme B (GzmB). This specific combination of antibodies allows for the identification of tumor-infiltrating Gr-1⁺/CD11b⁺ myeloid cells and NK1.1⁺, NK cells, cell types we have been implicated in gal-1-deficient tumor rejection. The immune infiltration profile of gal-1-deficient GL26-Cit glioma, referred to here as GL26-Cit-gal1i, is then compared to that of gliomas expressing normal levels of gal-1 called GL26-Cit-NT that contain a non-targeting control shRNA hairpin. The protocol begins with a description on how to culture GL26-Cit glioma cells in vitro, which is followed by an explanation on how to orthotopically engraft these cells into the striatum of syngeneic C57BL/6J mice. It then proceeds to enumerate the steps involved in the isolation and immunolabeling of glioma-infiltrating PBMCs for flow cytometric analysis. The protocol concludes with an explanation of standard data analysis and graphical representation.

The demonstration reveals that both Gr-1⁺/CD11b⁺ myeloid cells and NK1.1⁺ NK cells preferentially accumulate within the gal-1-deficient brain tumor microenvironment within 48 hr of tumor implantation, a result which helps explain why these tumors rapidly undergo complete tumor lysis approximately 1 week post-tumor engraftment²⁰. The method is easily adaptable to a number of different in vivo experimental designs in which temporal data on immune infiltration into the brain is required. A single experimentalist can perform the protocol from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in about 4-6 hr depending on the number of samples to be analyzed. The method may also be combined with experiments aimed to characterize the profile of circulating PBMCs in tumor bearing mice for comparison with those that infiltrate the brain so to identify immunosuppression phenotypes specifically induced by the tumor microenvironment. Application of this and similar methods should facilitate a better understanding of the factors involved in the trafficking of peripheral immune cells into the brain tumor microenvironment.

Protocol

Note: Please review the entire protocol prior to performing experiments. Approval for the use of vertebrate animals from the appropriate institutional committee on the use and welfare of animals must be obtained prior to proceeding.

1. Preparation of Tumor Cells for Intracranial Engraftment

1. Working in a class II biological safety cabinet, start by preparing GL26-Cit-NT/gal1i cell culture media by supplementing a 500 ml bottle of Dulbecco’s Modified Eagle Medium (DMEM) with 10% sterile-filtered heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 600 µg/ml G418 sulfate (for selection of the mCitrine expression vector) and 3 µg/ml of puromycin dihydrochloride (for selection of the non-targeting or gal-1-specific shRNAs).
2. Culture GL26-Cit-NT and/or GL26-Cit-gal1i cells (Figure 1A and 1B) in a tissue culture cabinet set to 37 °C and 5% CO₂ for 1-2 days prior to the tumor engraftment procedure or until the flasks reach 50-80% confluency.
3. On the day of surgery, remove the cell culture media from the glioma cells using a 10 ml serological pipette and a pipette gun and discard the media into a waste beaker.
   1. Invert the flask top-side-down and add 10 ml of DPBS to the topside of the flask using a 10 ml serological pipette. Slowly invert the flask to cover the cells in DPBS, then tip the flask vertically and remove and discard the DPBS into a waste beaker.
4. Add 3-4 ml of non-mammalian trypsin alternative in DPBS with EDTA (see materials list for details) to each T75 tissue culture flask and place back into the tissue culture cabinet for 2-5 min. Using a bright-field microscope, check that all of the cells have become detached from the bottom surface prior to proceeding.
5. Inhibit further enzymatic activity by diluting the trypsin alternative with 6 ml of DPBS. Pipette up and down using a 10 ml serological pipette to rinse the bottom surface of the flask and to mechanically dissociate any cellular aggregates. Aspirate the cells and place into respectively labeled 15 ml centrifuge tubes. Spin the cells down at 550 x g (max RCF) for 5 min at 4 °C (Figure 1C).
6. Remove the supernatant and gently resuspend the cells with 1 ml of un-supplemented DMEM. Make sure to pipette the cells up and down thoroughly with a P-1000 micropipette to achieve a single cell suspension.
7. Make a 1:10 dilution of the resultant cell suspension generated in 1.6 by placing 18 µl of un-supplemented DMEM into a 0.6 ml conical polypropylene microtube followed by the addition of 2 µl of the tumor cell suspension. Pipette up and down using a P-10 micropipette to homogenize the cells thoroughly.
8. Add 10 µl of the 1:10 tumor cell dilution generated in 1.7 to a separate 0.6 ml conical polypropylene microtube, then add 10 µl of Trypan blue stain to the tube. Thoroughly mix with a P-10 micropipette and add 10 µl of the resultant tumor cell/Trypan blue solution under a glass cover slip placed on top of a hemocytometer being careful not to introduce air bubbles (Figure 1D).
   1. Count the cells with a bright-field microscope using a 10X objective according to the specific protocol associated with the given hemocytometer. Be sure to factor in the 20-fold dilution of the original tumor cell suspension made during the preparation of the cells for accurate cell estimation in the original 1 ml aliquot. Use the following formula to calculate the total number of cells: total cells/ml = [((∑cells counted per square) / # of squares counted) x 20 (i.e., dilution factor) x 10,000 cells/ml].
9. After determining the total number of cells for each cell line used in the given experiment, spin them down at 550 x g (max RCF) for 5 min at 4 °C. Remove the supernatant(s) and resuspend the cells with an appropriate volume of un-supplemented DMEM in order to achieve a target concentration of 3 x 10⁴ cells per 1 µl for each cell line according to the following formula: (total # of cells / 30,000).
10. Place an equivalent volume of each cell line into respectively labeled 0.6 ml conical polypropylene tubes and place on ice (Figure 1E). Be sure to save some cells to continue propagating each cell line if separate T75s were not previously seeded.

2. Procedure for the Stereotactic Engraftment of Glioma Cells into the Striatum of C57BL/6J Mice

1. Prepare working solutions of surgical anesthesia, analgesia, and anesthesia reversal prior to surgery as follows:
   1. For ketamine/dexmedetomidine surgical anesthesia: remove 1.4 ml from a 10 ml vial of sterile 0.9% NaCl injection and replace with 600 µl of a 100 mg/ml stock solution of ketamine hydrochloride and 800 µl of a 0.5 mg/ml stock solution of dexmedetomidine hydrochloride to yield a mixed working solution of ketamine hydrochloride (6 mg/ml) and dexmedetomidine hydrochloride (0.04 mg/ml).
   2. For buprenorphine analgesia: remove 1 ml from a 10 ml vial of sterile 0.9% NaCl injection and replace with the entire 1 ml volume of a single 0.3 mg/ml stock buprenorphine hydrochloride ampul to yield a 0.03 mg/ml working solution.
   3. For atipamezole surgical anesthesia reversal: remove 1 ml from a 10 ml vial of sterile 0.9% NaCl injection and replace with 1 ml of a 5 mg/ml stock solution of atipamezole hydrochloride to yield a 0.5 mg/ml working solution.
2. Working in an approved location for mouse survival surgery, begin by setting up autoclaved or bead sterilized surgical instruments and other reagents as shown in (Figure 2A), then obtain enough 8-10 week-old female C57BL/6J mice (syngeneic with GL26 glioma) required for the study; ensure their continuous access to food and water.
3. Anesthetize the first mouse with a single intraperitoneal (i.p.) injection of the working anesthesia solution by delivering a dose of 75 mg/kg ketamine and 0.5 mg/kg dexmedetomidine (approximately 250 µl for a 20 g mouse). Next, give a 5 mg/kg subcutaneous (s.c.) injection of carprofen (approximately 100 µl for a 20 g mouse). Ensure that the mouse is non-responsive to toe and tail pinches prior to proceeding.
4. Using surgical clippers, shave the fur from the cranium. Apply povidone-iodine antiseptic solution to the shaved area, scrub with a 70% isopropyl alcohol prep pad, then re-apply povidone-iodine antiseptic solution. Next, apply a small amount of sterile petrolatum ophthalmic ointment to each eye to prevent drying.
5. Secure the skull in the stereotactic frame according to the following protocol:
   1. Carefully open the mouth by reaching in with a pair of curved dissecting forceps to gently pull out the tongue and move to one side of the mouth to prevent choking. Allow the forceps to retract while in the mouth to spread the jaw open and guide the top two incisors into the keyhole of the tooth bar on the stereotactic frame. Secure the tooth bar from pivoting horizontally or vertically by adjusting the respective screws. Ensure that the mouse’s cranium is level with the bench top.
   2. Place the ear bars securely against the postorbital bones of the cranium being careful not to apply too much inward pressure on the skull in order to prevent crushing.
   3. Place the nose bar over the snout and screw down until snug. Check that there is no vertical/lateral movement in the head by applying gentle pressure with a thumb and index finger. A mouse properly placed into a stereotactic frame is shown in (Figure 2B).
   4. Using a scalpel blade, make a 1 cm midline incision over the cranium from the frontal bone to the occipital bone. Retract the skin at the incision using Colibri retractors.
   5. Lower the microliter syringe equipped with a 33 G needle attached to the vertical arm of the stereotactic frame directly above the position of bregma (Figure 2C). From there, adjust the position of the needle +0.5 mm AP, +2.5 mm ML to reach the target site for tumor implantation. Use a 1 ml tuberculin syringe equipped with a 26 G needle to mark this location by gently excoriating the periosteum.
   6. Drill a hole into the cranium at the target site until reaching the underlying dura mater using a cordless precision power drill equipped with a 1/32” (0.8 mm) bit. While drilling, apply intermittent pressure followed by removal of the drill bit from the surface of the cranium to avoid excessive heat and force that may otherwise lead to the drill bit puncturing the underlying brain tissue.
6. Flush the microliter syringe with 0.9% NaCl in a 1.7 ml conical polypropylene microtube to ensure that the needle is not clogged. Gently flick the 0.6 ml conical polypropylene microtube containing the tumor cells several times to resuspend the cells.
7. Draw up 2 µl of cells into the microliter syringe ensuring that no air bubbles are introduced into the syringe. Dispense 1 µl of the cells onto a 70% isopropyl alcohol prep pad leaving exactly 1 µl of tumor cells remaining in the syringe.
8. Bring the needle down until it touches the dura mater, then introduce the needle into the brain -3.5 mm ventrally and retract by +0.5 mm. Slowly and smoothly deliver the cells by depressing the syringe plunger over the course of 1-2 min.
9. Allow the cells to settle at the injection site for 5 min prior to slowly withdrawing the needle. Wipe any coagulated blood or brain matter from the tip of the needle with a clean 70% isopropyl alcohol prep pad. Rinse the needle and syringe thoroughly with the 0.9% NaCl from step 2.6 to ensure that the needle is not clogged for the next injection.
10. Remove the Colibri retractors and suture the scalp using three 3-0 nylon monofilament sutures. Remove the mouse from the stereotactic frame and administer 0.1 mg/kg of buprenorphine hydrochloride subcutaneously (s.c.) (approximately 67 µl of the 0.03 mg/ml working solution for a 20 g mouse) followed by 2.5 mg/kg atipamezole hydrochloride intramuscularly (i.m.) (approximately 100 µl of the 0.5 mg/ml working solution for a 20 g mouse) for post-operative pain relief and anesthesia reversal. Place post-surgical mice back into their cages under a heating lamp to recover.

3. Mouse Transcardial Perfusion and Harvesting of the Brain

1. Prepare heparinized Tyrode’s Solution according to the following protocol:
   1. Put a magnetic stirring bar into a 1 L glass screw-cap storage bottle and fill to volume with ultrapure deionized water. Place the bottle on a stirring plate.
   2. Weigh and add the following chemicals to the glass bottle while stirring: 8 g sodium chloride (NaCl), 0.264 g calcium chloride (CaCl₂•2H₂O), 0.05 g Sodium phosphate monobasic (NaH₂PO₄•2H₂O), 1.0 g D-glucose (C₆H₁₂O₆), 1.0 g sodium bicarbonate (NaHCO₃), 0.2 g potassium chloride (KCl). Allow the salts to dissolve, then add 100 µl of a 1,000U/ml stock solution of heparin sodium to the Tyrode’s solution.
   3. Remove the container from the stirring plate and extract the stirring bar using a magnetic wand. Store heparinized Tyrode’s solution at 4 °C.
2. Prepare a working solution for terminal anesthesia as follows:
   1. For ketamine/xylazine terminal anesthesia: remove 1,360 µl from a 10 ml vial of sterile 0.9% NaCl injection and replace with 1,200 µl of a 100 mg/ml stock solution of ketamine hydrochloride and 160 µl of a 100 mg/ml stock solution of xylazine hydrochloride to yield a mixed working solution of ketamine hydrochloride (12 mg/ml) and xylazine hydrochloride (1.6 mg/ml).
3. Prepare density centrifugation media mix solution by placing 90 ml of 10x PBS into 264 ml of ultrapure deionized water (3.93x) in a sterile plastic container. Titrate to pH 7.0-7.2 with HCl and filter sterilize through a 0.22 µm filter. Store at 4 °C.
4. Prepare 70% density centrifugation media by combining 18 ml of density centrifugation media mix solution with 30 ml of density centrifugation media (see materials list for details) in a 50 ml polypropylene centrifuge tube. Store at 4 °C, but bring to RT before use.
5. Prepare 37% density centrifugation media by combining 9.6 ml of DPBS with 10.4 ml 70% density centrifugation media in a 50 ml centrifuge tube. Store at 4 °C, but bring to RT before use.
6. Prepare flow buffer by placing 500 µl of FBS into 50 ml of DPBS (~1% FBS) in a 50 ml centrifuge tube. Store at store at 4 °C
7. Fill a 4.5 L polyurethane ice bucket to capacity with ice chips.
8. Make a combined solution of 1 mg/ml collagenase and 1 mg/ml DNase-I in sufficient quantity for 1 ml per brain sample in the study by diluting a 10 mg/ml DNase-I stock solution 1:10 and a 50 mg/ml collagenase stock solution 1:50 with sterile DPBS in a single 15 ml polypropylene centrifuge tube. Gently mix the solution by pipetting up and down with a P-1000 micropipette. Store on ice in the polyurethane bucket from step 3.7.
9. Obtain two 7 ml glass Dounce tissue grinders. Label one “NT” and the other “gal1i”. Place the tissue grinders in the ice bucket and add 1 ml of DPBS to each.
10. Obtain enough 15 ml centrifuge tubes to collect brain matter from each mouse in the study and place in the ice bucket.
11. Place ~150 ml of DPBS among three 50 ml centrifuge tubes into the ice bucket.
12. Obtain enough polystyrene hexagonal weighing dishes (top inner diameter (I.D.) 115 mm, base I.D. 85 mm, 203 ml volume) for each mouse in the study. The complete set up for dissection and trituration of mouse brains is shown in (Figure 3A)
13. At chosen time points, anesthetize the first tumor-bearing mouse in the study using a single i.p. injection of 150 mg/kg of ketamine hydrochloride and 20 mg/kg xylazine hydrochloride (approximately 250 µl of the combined 12 mg/ml ketamine/1.6 mg/ml xylazine working solution for a 20 g mouse) to induce deep anesthesia. Ensure that the mouse is non-response to toe and tail pinches prior to proceeding.
14. Retrieve the previously prepared Tyrode’s solution and place into a laminar flow hood dedicated to terminal animal surgical procedures. Place gas impermeable polymeric tubing attached to a carbogen tank into the Tyrode’s solution. Open the tank valve to allow 95% O₂/5% CO₂ carbogen to continuously bubble into the solution.
15. Attach a 20 G aluminum hub blunt needle to the end of additional gas impermeable polymeric tubing attached to a peristaltic pump. Place the tubing at the other end of the peristaltic pump into the Tyrode’s solution and turn the pump on to allow the tubing to fill with oxygenated and heparinized Tyrode’s solution. The set up for mouse transcardial perfusion is shown in (Figure 3B).
16. Using four 26 G needles, pin down the anesthetized mouse ventral side up by the front and hind paws on an extruded polystyrene foam block covered with a disposal absorbent towel in the laminar flow hood.
17. Grasp the skin above the peritoneal cavity using a pair of blunt dissection forceps, and using a large pair of dissection scissors make a “Y” incision by penetrating the peritoneal wall, cutting cephalically to puncture the diaphragm, then bifurcating at the sternum to terminate at the left and right axillae.
18. Use a hemostat to clamp the sternum and reflect the rib cage over the mouse’s left shoulder. Remove the pericardium with the pair of blunt dissection forceps.
19. Turn on the peristaltic pump to begin a steady drip of oxygenated and heparinized Tyrode’s solution (roughly 2.3-2.5 ml/min).
20. Insert the 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 (Figure 3C).
21. Allow the Tyrode’s solution to thoroughly perfuse the mouse circulatory system until the liver and lungs have completely blanched due to the lack of blood. Ensure that no gross amounts of blood continue to exit the right atrium prior to removing the 20 G aluminum hub blunt needle from the left ventricle.
22. 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 at the level of the neck.
23. Using a small pair of dissection scissors, cut the scalp at the midline beginning at the occipital bone working forward towards the snout in order to expose the cranium.
24. Retract the skin on both sides of the cranium using a thumb and index finger and hold the skull securely. Use a pair of bone Rongeurs to break through the cranium beginning at the occipital bone and work forward to completely expose the dorsal and lateral surfaces of the brain. Be careful to minimize damage to the brain.
25. Once the cranial bones have been removed, turn the head of the mouse ventral side up and use a small pair of dissection scissors to cut the cranial nerves at the base of the brain in order to liberate it from the skull.

4. Isolation of Glioma-infiltrating PBMCs

1. 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 bisect the two hemispheres. Turn the ipsilateral hemisphere medial side down and make two coronal cuts at the levels of the cerebellum and the olfactory bulb to isolate the target tissue containing the tumor implant (Figure 4A). An equivalent portion of the contralateral hemisphere may also be isolated and used as a negative control.
2. Place the target tissue into the respectively labeled glass Dounce tissue grinder containing 1 ml of DPBS, push the plunger all the way down and twist 7 times to initially disrupt the tissue. 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 4 times during the next two repeats and 3 times during the last repeat to avoid over triturating the tissue.
3. Using a P-1000 micropipette, sequentially apply three 1 ml volumes of ice-cold DPBS (prepared in 3.11) along the sides of the plunger to rinse into the tissue grinder.
4. Resuspend the triturated brain matter by pipetting up and down and place into a labeled 15 ml centrifuge tube on ice. Rinse the sides of the Dounce tissue grinder with 1 additional ml of ice-cold DPBS and add to the same 15 ml centrifuge tube. Keep all tubes containing triturated brain matter on ice until all samples have been processed.
5. Repeat steps 3.13-3.25 and 4.1-4.4 for each mouse in the study.
6. Once all samples have been processed, spin down the triturated brain matter in the 15 ml centrifuge tubes at 740 x g (max RCF) for 20 min at 4 °C.
7. Remove the supernatant with a 10 ml serological pipette and pipette gun and resuspend the pelleted brain matter in 1 ml of the previously prepared collagenase/DNase-I digestive enzymes using a P-1000. Place the 15 ml centrifuge tubes into a test tube rack and place in a 37 °C water bath for 15 min.
8. Gently agitate the samples twice throughout the incubation period by flicking the tubes to facilitate tissue disaggregation.
9. Add 6 ml of ice-cold DPBS using a 10 ml serological pipette to each tube to dilute the digestive enzymes. Pipette up and down to resuspend, and filter the total volumes through sterile 70 µm nylon mesh filters into new labeled 15 ml centrifuge tubes on ice.
10. Spin the brain cell suspension down at 740 x g (max RCF) for 20 min at 4 °C to achieve a single cell pellet (Figure 4B). After removing the 15 ml tubes from the centrifuge place them on ice and increase the temperature setting on the centrifuge to about 21 °C in preparation for the next step.
11. Fully remove the supernatant from each tube containing brain cells and initially resuspend the pellets in 1 ml of 70% density centrifugation media using a P-1000 micropipette. Then add 4 additional milliliters of 70% density centrifugation media to each tube using a 10 ml serological pipette. Screw the caps on securely and homogenize the cell suspension by gently inverting to tubes several times.
12. Remove the 15 ml centrifuge tubes containing brain cells resuspended in 70% density centrifugation media from ice, and place in a test tube rack at RT. One at a time, carefully overlay 2 ml of 37% density centrifugation media solution onto the 5 ml of 70% density centrifugation media with a P-1000 micropipette to form a clean interface between the two density centrifugation media layers (Figure 4C).
    1. Use a fine-tipped marker to indicate the position of the interface so it can be easily identified after centrifugation, when the distinction becomes less apparent.
13. Spin down the 15 ml centrifuge tubes at 740 x g (max RCF) for 20 min at RT with no break to avoid disrupting the interface.
14. After centrifugation, collect the PBMCs that have accumulated at the interface between the two density centrifugation media layers (Figure 4D) by introducing a P-200 micropipette into the tube along its side, carefully bypassing the lipid layer.
    1. Once at the level of the PBMC band, slowly extract 200 µl from the surface of the 70% density centrifugation media layer and place into a respectively labeled polypropylene FACS tube on ice. Repeat once for a total volume of 400 µl per sample.
15. Add 3 ml of flow buffer to each FACS tube containing 400 µl of PBMCs to sufficiently dilute the density centrifugation media, then spin the cells down at 660 x g (max RCF) for 20 min at 4 °C.

5. Immunolabeling of Glioma-infiltrating PBMCs

1. Prior to analyzing any experimental PBMC samples, perform a single, independent, cell surface isotype control experiment to establish baseline gates to be associated with a set of fixed PMT voltages within a flow cytometric data analysis template dedicated to the assessment of glioma-infiltrating PBMCs according to the following protocol:
   1. Engraft one C57BL/6J mouse with GL26-Cit-gal1i and another with GL26-Cit-NT according to the procedure outlined in section 2. After 72 hr of tumor growth, euthanize both mice and isolate the tumor-infiltrating PBMCs according to the procedures outlined throughout sections 3 and 4.
   2. Combine the two PBMC samples (NT and gal1i) into one volume (i.e., 100 µl), then further divide the sample evenly among three FACS tubes (i.e., 33.3 µl per tube) labeled “CD45 isotype”, “Gr-1/CD11b isotype”, and “NK1.1 isotype”. Add the following antibodies (each at a 1:100 dilution and diluted to a total volume of 200 µl in flow buffer) to the respectively labeled FACS tubes: “CD45 isotype”: Alexa Fluor 700-conjugated rat IgG2b, κ (clone: RTK4530); “Gr-1/CD11b isotype”: Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11), PE-conjugated rat IgG2b, κ (clone: eB149/10H5), and PerCP/Cy5.5-conjugated rat IgG2b, κ (clone: RTK4530); “NK1.1 isotype”: Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11), PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5), PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70), and APC-conjugated mouse IgG2a, κ (clone: eBM2a).
   3. Allow PBMCs to immunolabel on ice in the dark for 20 min. Flick the tubes once halfway through the incubation period to gently mix. Wash with 1 ml of flow buffer, spin down at 660 x g (max RCF) for 10 min at 4 °C and resuspend each FACS tube containing PBMCs with 200 µl of flow buffer.
   4. Using a flow cytometer equipped with an 85 µm integrated nozzle, analyze the “CD45 isotype“ tube first to establish a baseline CD45 interval gate. Next, run the “Gr-1/CD11b isotype” tube to establish a baseline Gr-1/CD11b quadrant gate using only true CD45⁺ cells as input. Finally, run the “NK1.1 isotype” tube to establish a baseline NK1.1 interval gate using only true CD45⁺/Gr-1^low/CD11b^+/- cells as input.
2. Use the following procedure for all future experimental analysis:
   1. Make a sufficient quantity of a 1:100 dilution of cell surface antibodies in flow buffer to achieve a 200 µl volume per sample (plus 1 for the GzmB isotype): Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11), PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5), PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70), APC-conjugated mouse anti-mouse NK1.1 (clone: PK136).
   2. Carefully remove the supernatant of the spun down PBMCs from 4.15 until the meniscus is just above the bottom of each FACS tube (~50 µl) to avoid cell losses. Resuspend the PBMCs in each FACS tube using the residual flow buffer with a P-200 micropipette and remove (1/# of samples) worth of the volume from each FACS tube and combine into a new FACS tube labeled “pooled isotype”.
   3. Add 200 µl of the cell surface antibody cocktail prepared in 5.2.1 to each PBMC sample. Allow PBMCs to immunolabel on ice in the dark for 20 min. Flick the tubes once halfway through the incubation period to gently mix.
   4. Wash with 1 ml of flow buffer; spin down at 660 x g (max RCF) x 10 min at 4 °C.
   5. Resuspend all FACS tubes containing PBMCs in 200 µl of a commercially available formalin-based fixative (see materials list for details) for 15 min on ice in the dark. Flick the tubes once halfway through the incubation period to gently mix.
   6. Wash with 1 ml of a commercially available 1x permeabilization/wash buffer (see materials list for details) and spin down at 660 x g (max RCF) for 10 min at 4 °C.
   7. While PBMCs are spinning down, prepare a sufficient quantity of a 1:100 dilution of Pacific Blue-conjugated mouse anti-mouse GzmB (Clone: GB11) intracellular antibodies in 1x permeabilization/wash buffer to achieve a 200 µl volume per sample.
   8. In a separate tube, prepare a single 200 µl volume of a 1:100 dilution of Pacific Blue-conjugated mouse IgG1, κ (clone: MOPC-21) isotype control antibody.
   9. Resuspend each of the experimental PBMC samples with 200 µl of the 1:100 dilution of anti-mouse GzmB antibodies and resuspend the single FACS tube labeled “pooled isotype” with the 200 µl volume of the 1:100 dilution of isotype antibodies.
   10. Allow all PBMC samples to immunolabel on ice in the dark for 20 min. Flick the tubes once halfway through the incubation period to gently mix.
   11. Wash with 1 ml of flow buffer; spin down at 660 x g (max RCF) for 10 min at 4 °C and resuspend PBMCs with 200 µl of flow buffer for analysis.
   12. Flow cytometrically analyze glioma-infiltrating PBMCs using an 85 µm integrated nozzle and the gates and PMT voltages previously established in section 5.1²². Be sure to run the entire volume of each sample on the flow cytometer to ensure a fair comparison of the total number of glioma-infiltrating PBMCs in each sample.

Results

The following gating strategy is used for a typical experiment: FSC-A vs. SSC-A → SSC-H vs. SSC-W → FSC-H vs. FSC-W → CD45 vs. count → Gr-1 vs. CD11b → NK1.1 vs. count. Gates placed on Gr-1⁺/CD11b⁺ myeloid cells and NK1.1⁺ NK cells are then stratified based on GzmB expression (Figure 5A). Backgating those cells identified as NK1.1⁺ NK cells and Gr-1⁺/CD11b⁺ myeloid cells in our experiments onto FSC-A vs. SSC-A confirms the smaller lymphoid...

Discussion

This protocol describes a robust and reproducible method for the isolation and flow cytometric analysis of PBMCs that have infiltrated the early mouse brain tumor microenvironment. Glioma cell suspensions are generated at a concentration specified by the experimentalist that are stereotactically engrafted into the striatum of the mouse brain. Mice are then euthanized at predetermined time points specified by the experimental design and their brains are harvested and processed to isolate glioma-infiltrating PBMCs which ar...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco’s Modification of Eagles Medium	Gibco	12430-054	with 4.5g/L D-glucose, L-glutamine, 25 mM HEPES and phenol red
Dulbecco’s Phosphate-buffered Saline	Gibco	14190-144	without calcium chloride or Magnesium chloride
Fetal bovine serum	Omega Scientific Inc.	FB-11
Penicillin Streptomycin	Gibco	15140-122	10,000 U/ml Penicillin; 10,000 μg/ml Streptomycin
L-glutamine	Gibco	25030-081	200 mM
G418 sulfate	Omega Scientific Inc.	GN-04
Puromycin dihydrochloride	Sigma-Aldrich	P8833	from Streptomyces alboniger
HyClone HyQTase non-mammalian trypsin alternative	Thermo Scientific	SV30030.01	in DPBS with EDTA
Phase contrast hemocytometer	Sigma-Aldrich	Z359629	0.1 mm deep
Trypan blue stain	Gibco	15250-061
Sterile 0.9% NaCl injection, USP	Hospira	NDC: 0409-4888	10 ml vials
0.6 ml conical polypropylenemi microtubes	Genesee Scientific	22-272
Atipamezole hydrochloride injection	Orion Pharma	NDC: 52483-6298	5 mg/ml solution
Ketamine hydrochloride injection	Fort Dodge	NDC: 0409-2051	100 mg/ml solution
Dexmedetomidine hydrochloride injection	Zoetis	NDC: 54771-2805	0.5 mg/ml solution
Xylazine hydrochloride injection	Lloyd	NDC: 61311-481	100 mg/ml solution
Carprofen injection	Pfizer	NDC: 61106-8501	50 mg/ml solution
Buprenorphine hydrochloride injection	Reckitt Benckiser	NDC: 12496-0757	0.3 mg/ml ampuls
1 ml tuberculin syringes	Covidien	8881501400
26G x ½ (0.45 mm x 13 mm) syringe needles	BD	305111
Surgical clippers	3M	9661
Providone-iodine solution	Aplicare	82-217	NDC: 52380-1905
Sterile petrolatum ophthalmic ointment	Dechra	NDC: 17033-211
70% isopropyl alcohol prep pads	Kendall	6818
Sterile gauze	Covidien	8044	non-woven, 4" x 4", 4-ply
1.7 ml conical polypropylene microtubes	Genesee Scientific	22-281
Mouse stereotactic frame	Stoelting	51730
Surgical lamp	Philips Burton	CS316W	Coolspot II Variable Spotlight
Curved dissecting forceps	Ted Pella Inc.	5431
Colibri retractors	FST	17000-03
Cordless precision power drill	Dremel	1100-01	Stylus model; 7.2-Volt Lithium-Ion with Docking Station
Engraving Cutter	Dremel	105	1/32" or 0.8 mm bit diameter
Microliter syringe	Hamilton	75	Hamilton Microliter Syringes 700 series; 5 μl volume
Microliter syringe needles	Hamilton	7762-06	33 G, small hub RN NDL, 1.5 in, point style 3, 6/PK
Ethilon 3-0 black monofilament nylon sutures	Ethicon	1663
Magnetic stir bar	VWR	74950-296	7.9 mm diameter x 50 mm length (5/16" diameter x 2" length)
1 L glass screw-cap storage bottle	Corning	1395	Type 1, Class A borosilicate glass
Heparin sodium	Sagent	NDC: 25021-400	1,000 U/ml solution
Peristaltic pump	Cole Parmer Instruments Group	77200-60	Master Flex Easy Load II console drive
compressed carbogen (95% O2 / 5% CO2)	available from local vendor	N/A	A-type, large cylinder
20 G x 1 ½" aluminum hub blunt needles	Kendall	8881202363	0.9 mm x 38.1 mm
Polyurethane ice bucket	Fisherbrand	02-591-45	Capacity: 0.152 oz. (4.5L)
Collagenase (Type I-S)	Sigma Aldrich	C1639	from Clostridium histolyticum
Deoxyribonuclease I	Worthington Biochemical Corp.	LS002007	>2,000 Kunitz units per mg dry weight
Antistatic polystyrene hexagonal weighing dishes	Ted Pella Inc.	20157-3	top I.D. 115 mm, base I.D. 85 mm, 203 ml volume
Stainless steel single edged razor blades	Garvey	40475
Bone rongeurs	FST	16001-15
Hemostat	FST	13014-14
Large dissection scissors	Ted Pella Inc.	1316
Small dissection scissors	FST	14094-11
Blunt end forceps	Ted Pella Inc.	13250
7 ml glass Dounce tissue grinder	Kontes	KT885300-0007
Percoll Density Centrifugation Media	GE Healthcare	GE17-0891-01
10 ml serological pipettes	Genesee Scientific	12-104	single use; sterile
70 μm sterile nylon mesh cell strainers	Fisherbrand	22363548
Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)	Biolegend	103128
APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)	eBiosciences	17-5941-82
PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)	BD Pharmigen	553128
PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)	Biolegend	101228
Alexa Fluor 700-conjugated rat IgG2b, κ (clone: RTK4530) isotype control	Biolegend	400628
APC-conjugated mouse IgG2a, κ (clone: eBM2a) isotype control	eBioscience	17-4724
PE-conjugated rat IgG2b, κ (clone: eB149/10H5) isotype control	eBioscience	12-4031-82
PerCP/Cy5.5-conjugated rat IgG2b, κ (clone: RTK4530) isotype control	Biolegend	400632
Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)	Biolegend	515403
Pacific Blue-conjugated mouse IgG1, κ (Clone: MOPC-21) isotype control	Biolegend	400151
Cytofix/Cytoperm	BD	554714
15 ml polypropylene centrifuge tubes	Genesee Scientific	21-103	conical bottom
50 ml polypropylene centrifuge tubes	Genesee Scientific	21-108	conical bottom
12 mm x 75 mm round-bottom polypropylene FACS tubes	Fisherbrand	14-956-1B
FACSAria Special Order Research Product flow cytometer/cell sorter	BD	650033
Class II Biological Safety Cabinet	The Baker Company	SG603	model: SterilGARD III Advance