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.
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.
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-tumorigenic6,7. 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 (Treg) 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 immunotherapeutics13-19. 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.
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
2. Procedure for the Stereotactic Engraftment of Glioma Cells into the Striatum of C57BL/6J Mice
3. Mouse Transcardial Perfusion and Harvesting of the Brain
4. Isolation of Glioma-infiltrating PBMCs
5. Immunolabeling of Glioma-infiltrating PBMCs
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 size of NK cells and the relatively large size of myeloid cells (Figure 5B). Raw data files (i.e., fcs files) are analyzed by commercially available flow cytometric data analysis software such as Flowjo.
A total of 18 C57BL/6J mice were used to demonstrate this method. Nine (9) were engrafted with GL26-Cit-NT and 9 were engrafted with GL26-Cit-gal1i on the same day using equivalent number of cells (3×104). Three mice from each group were euthanized at 24, 48, and 72 hr post-tumor engraftment. The data show that engraftment of GL26-Cit-gal1i glioma cells into the brain of syngeneic C57BL/6J mice rapidly induces the recruitment of CD45+ PBMCs (Figure 6A). Based on the specific antibody cocktail used in the demonstration it can further be shown that Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells specifically enter the gal-1-deficient tumor microenvironment within 48 hr of tumor engraftment (Figure 6B and 6C); however the total number of glioma-infiltrating myeloid cells far outweighs that of NK cells.
Figure 1: Preparation of GL26-Cit Cells for Intracranial Engraftment. (A) Representative 10X bright-field and epifluorescence micrographs of GL26-Cit-NT (left images) and GL26-Cit-gal1i (right images) cells grown in culture. (B) Western blot of GL26-Cit-NT (left lane) and GL26-Cit-gal1i (right lane) whole cell lysate. Gamma tubulin (γ-tub.) is shown as a loading control. (C) GL26-Cit cell pellet from an approximately 50% confluent T75 tissue culture flask after centrifugation at 550 x g (max RCF) for 5 min at 4 °C. (D) Bright-field view of a hemocytometer containing GL26-Cit cells (white dots) diluted 1:1 with Trypan Blue to assess cell number and viability. Cells should be >90% viable to ensure reproducible tumor growth. The average of the cell counts from the squares labeled 1 through 5 should be taken to increase the accuracy of cell count estimation. (E) GL26-Cit cells resuspended at 3 x 104 cells/μl in un-supplemented DMEM for intracranial implantation. Please click here to view a larger version of this figure.
Figure 2: Stereotactic Engraftment of Glioma Cells into the Striatum of C57BL/6J Mice. (A) Surgical suite layout showing the required surgical instruments and reagents. (B) Close up view of a C57BL/6J mouse harnessed in a stereotactic frame with proper needle placement at +0.5 mm AP, +2.5 mm ML, and -3.0 mm DV relative to bregma. (C) Dorsal view of the mouse skull showing the position of bregma and the target site for tumor cell engraftment. Please click here to view a larger version of this figure.
Figure 3: Mouse Transcardial Perfusion. (A) Required reagents for dissection, trituration, and enzymatic digestion of mouse brain tissue. (B) Layout of the instruments and reagents required for mouse transcardial perfusion with oxygenated and heparinized Tyrodes’s solution shown in a laminar flow hood dedicated to terminal mouse surgical procedures. (C) Schematic representation of the heart demonstrating proper instruments and placements required for transcardial perfusion. Please click here to view a larger version of this figure.
Figure 4: Isolation of Glioma-infiltrating PBMCs. (A) Strategy for the dissection of mouse brain tissue containing early stage orthotopic glioma implants. The top panel demonstrates the sagittal bisection of the ipsilateral hemisphere away from the contralateral hemisphere. The bottom panel demonstrates the two additional coronal cuts necessary to isolate the target tissue containing the tumor implant (highlighted in purple). (B) Single cell brain tissue pellet (white arrow) after enzymatic digestion, filtration through a 70 μm nylon mesh and centrifugation. (C) Properly poured density centrifugation media gradient prior to centrifugation. The white arrow indicates the clean interface formed between the two density centrifugation media layers. (D) Density centrifugation media gradient after centrifugation demonstrating the lipid layer that forms at the top of the 37% density centrifugation media layer white arrow) and the PBMC band (outlined by the dashed black lines) at the interface between the two layers. Please click here to view a larger version of this figure.
Figure 5: Flow Cytometric Gating Strategy Used to Identify Glioma-infiltrating Gr-1+/CD11b+ Myeloid Cells and NK Cells. (A) Step 1: Total immunolabeled cells isolated from the PBMC band of a density centrifugation media gradient are gated on to exclude cellular debris (FSC-A less than ~50 K). Steps 2 and 3: Doublet discrimination gating to filter out cellular aggregates. Step 4: CD45 gate to identify glioma-infiltrating immune cells. Isotype control is shown as the grey silhouette. Step 5: CD45+ cells stratified based on Gr-1 and CD11b. Isotype control for both Gr-1 and CD11b is overlaid on the plot and enclosed by the gold circle. The red circle indicates Gr-1+/CD11b+ myeloid cells. Step 5’: Gr-1+/CD11b+ myeloid cells stratified based on GzmB expression. As predicated, these cells do not label with anti-GzmB antibodies over isotype control (grey silhouette). Step 6: Gr-1low cells outlined by the black rectangle in Step 5 stratified based on NK1.1 expression. Isotype control is shown as the grey silhouette. Step 6’: NK1.1high cells stratified based on GzmB expression. These cells indeed label with anti-GzmB antibodies above isotype control (grey silhouette). (B) Backgating of Gr-1+/CD11b+ myeloid cells onto FSC-A vs. SSC-A demonstrating that NK cells have a smaller lymphoid size as compared to the larger sized Gr-1+/CD11b+ myeloid cells. Please click here to view a larger version of this figure.
Figure 6: Comparison of PBMC Infiltration into the GL26-Cit-NT vs. GL26-Cit-gal1i Tumor Microenvironment over the First Three Days of Intracranial Tumor Growth. (A) Total CD45+ immune cells. (B) CD45+/Gr-1+/CD11b+ myeloid cells. (C) CD45+/NK1.1+ NK cells. Data points from PBMCs isolated from GL26-Cit-gal1i gliomas are connected by smooth lines, whiles those isolated from GL26-Cit-NT gliomas are connected by dashed lines. Glioma-infiltrating PBMCs from three mice were analyzed per tumor type at each time point. Numbers associated with each data point on the GL26-Cit-gal1i curves represent the fold-induction of that particular PBMC type over GL26-Cit-NT at the specified hour post-tumor implantation (HPI). Data represent the mean number of immune cells counted ± the standard error of the mean (SEM). Statistical analysis was performed by 2-way analysis of variance. Please click here to view a larger version of this figure.
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 are immunolabeled with combinations of fluorochrome-conjugated primary antibodies; immunolabeled cells are then quantified by flow cytometry. Although the demonstration presented here focuses on early time points, glioma-infiltrating PBMCs can be assessed at any time point post-tumor engraftment up until mouse moribundity. The method is highly modular as any number of different antibody combinations may be used to examine immune cells of interest including T cells, B cells, NK cells, monocytes, and macrophages. Note that the protocol has been optimized for use with female C57BL/6J mice 8-10 weeks of age. Mice outside of this age range may have altered percentages of circulating PBMCs, which may lead to cell counts at equivalent time points that are not representative of the data presented here. Characterization experiments may also be required if alternative tumor models are used or if mice other than those on the B6 background are used due to the fact that strains can differ in their PBMC profiles (Jaxpheno6; Mouse Phenome Database; the Jackson Laboratory). Gender and environmental conditions can also be sources of disparity among PBMC frequencies and percentages.
This method has been demonstrated using a combination of four cell surface antibodies that enable the detection of Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells, cell types implicated in the rejection of gal-1-deficient glioma. The inclusion of intracellular GzmB antibodies further enables the detection of cytotoxic potential in the NK cell subset. Representative results are provided for immune cell infiltration into early GL26-Cit gliomas in syngeneic C57BL/6J mice that either express normal levels of gal-1 or which have reduced levels due to shRNA-mediated gene knockdown. The demonstration exhibits the sensitivity and reproducibility of the technique by showing that glioma-infiltrating PBMCs can be reproducibly isolated and quantified as soon as 24 hr post-tumor engraftment. Aside from revealing a population of tumor-infiltrating Gr-1high/CD11b+ myeloid cells, the demonstration also reveals a population of Gr-1int./CD11b+ cells whose identity is currently undefined; additional experiments are necessary to further decipher the character of this cell population. We also note that we do not observe a readily distinguishable population of CD45int/Gr-1–/CD11bhigh/NK1.1– cells consistent with microglia as previously described by others23,24. A simple explanation for this result is that the specific isolation protocol used here precludes their accumulation at the 37/70 density centrifugation media interface. It is also important to point out that the cells collected from the 37/70 density centrifugation interface do not appear to include the glioma cells themselves. This is evident by the fact that GL26-Cit glioma cells, which appear between 100-200K on SSC-A and 100K of FSC-A using equivalent PMT voltages to those used in this demonstration (data not shown) are absent from the FSC-A vs. SSC-A plots (refer to Figure 5A; Step 1).
Anti-Gr-1 antibodies recognize both the Ly6G and Ly6C cell surface proteins that are specific to polymorphonuclear and mononuclear myeloid cells, respectively25. The use of anti-Gr-1 antibodies thus precludes distinction between these two cell populations. Preliminary results in our lab are now beginning to shed light on a more accurate description of the early Gr-1+/CD11b+ cells that infiltrate the gal-1-deficient glioma microenvironment. Experiments incorporating anti-Ly6G (clone: 1A8) and anti-Ly6C (clone: AL-21) antibodies together with anti-CCR2 antibodies now indicate that the Gr-1high/CD11b+ cells infiltrating the early gal-1-deficient tumor microenvironment are consistent with Ly6Chigh/CCR2high inflammatory monocytes, although further experiments are required to confirm this result (data not shown).
Due to cell loses that may occur during the repeat centrifugation and resuspension steps involved in isolating glioma-infiltrating PBMCs, it is likely that observed cell counts are in fact lower that what are actually present in the intact brain. However, differences between experimental groups should hold if equivalent volumes of the 37/70 density centrifugation media interface are extracted and if the entire volume of each sample is analyzed by the flow cytometer. Failure to adhere to these steps will result in unfair comparisons between samples and will preclude unbiased analysis. The inability to quantify the exact number of PBMCs entering the brain tumor microenvironment is not a limitation specific to this protocol. Alternative methods such as immunohistological cell counting strategies are also subject to error due to extrapolations of total cell numbers based on stereological counts and the requirement of equivalent image thresholding on tissue section micrographs, which may differ in their level of background staining, potentially leading to false-negative or false-positive signals. Nevertheless, a comparison of time course analysis between different analytical methods should reveal time-dependent immune-influx curves with similar overall shape. An additional drawback to immunohistochemical methods is the inability of certain antibodies validated for flow cytometry to bind to the equivalent antigenic epitope in the context of formalin-fixed brain tissue sections.
There are a few key steps in this protocol that may serve as limitations for those unfamiliar with its methodology. One such step is the harvesting of the brain from the cranium without damaging it. We suggest practicing the technique several times prior to running proper experiments. However, since the brain will eventually be triturated, minor damage to the superficial layers of the brain are likely inconsequential to the accurate quantification of glioma-infiltrating PBMCs. A second step that inexperienced investigators may initially find difficult is the pouring of the density centrifugation media gradient. The experimentalists’ ability to form a clean interface while overlying the 2 ml of 37% density centrifugation media on top of the 70% layer is crucial to reproducible isolation of PBMCs. Although this step may initially prove challenging, it vastly enriches for PBMCs and dramatically decreases the period of time otherwise required for sample analysis if whole brain tissue where to be analyzed flow cytometrically. PBMC enrichment also reduces the total number of events captured by the flow cytometer, thus reducing .fcs file sizes and helping to resolve rare brain-infiltrating immune cell populations. For best results in establishing a clean density centrifugation interface we recommend pouring the 37% density centrifugation media using a P-1000 micropipette with smooth plunger action. Angle the 15 ml centrifuge tube approximately 20-30° above the bench top. Rest the tip of the micropipette on the side of the tube 3-5 ml markings above the surface of the 70% density centrifugation media layer. Pour steadily and slowly so to minimize overt outward momentum of the 37% density centrifugation media as it extrudes from the micropipette tip in order to avoid disturbing the underlying 70% density centrifugation media. Lastly, the fact that the brain tissue architecture is necessarily destroyed in the process of isolating glioma-infiltrating PBMCs means that additional mice will be required to obtain time point matched histological data.
Newer glioma models generated through the injection of oncogenic plasmid DNA into the normal rodent brain have been developed in recent years26-32. These “endogenous” tumor models circumvent potential artifacts caused by stereotactically engrafting ex vivo cancer cells and more closely mimic the histological hallmarks of clinical glioma. The method described herein is well suited to the study of immune infiltration events that characterize these more clinically relevant model systems. Studies on immune influx in neonatal mice may also be performed using this method although the density of neonatal mouse brain is less than that of adults, which may require an optimization of the enzymatic digestion step in order to prevent over digestion of the brain tissue. Additional applications of the technique include investigations on alternative classes of inflammatory brain disease aside from malignant tumors such as experimental allergic encephalomyelitis (EAE) and immune responses to viral infection.
The authors have nothing to disclose.
This work was supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) grants R01-NS074387, R01-NS057711 and R21-NS091555 to M.G.C.; NIH/NINDS grants R01-NS061107, R01-NS076991, R01-NS082311 and R21-NS084275 to P.R.L.; grants from Leah’s Happy Hearts, University of Michigan Comprehensive Cancer Center awarded to M.G.C. and P.R.L; the Department of Neurosurgery, University of Michigan School of Medicine; the Michigan Institute for Clinical and Health Research, supported by NIH grant 2UL1-TR000433; the University of Michigan Cancer Biology Training Grant supported by NIH/NCI (National Cancer Institute) grant T32-CA009676; the University of Michigan Training in Clinical and Basic Neuroscience supported by NIH/NINDS grant T32-NS007222; and the University of Michigan Medical Scientist Training Program supported by NIH/NIGMS (National Institute of General Medicine Sciences) grant T32-GM007863. The authors are thankful for the academic leadership and support received from Dr. Karin Muraszko and the Department of Neurosurgery; to M. Dahlgren, D. Tomford, and S. Napolitan for superb administrative support; to M. Dzaman for outstanding technical assistance; and to Phil F. Jenkins for generous support toward the purchase of a Zeiss 3D Scanning Electron Microscope. We also acknowledge the Kuchroo laboratory at Harvard Medical School from which a modified version of the density centrifugation media-mediated strategy for isolation of brain mononuclear cells was drawn.
Dulbecco’s Modification of Eagles Medium | Gibco | 12430-054 | with 4.5g/L D-glucose, L-glutamine, 25mM 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 | 10,000U/ml Penicillin; 10,000μg/ml Streptomycin |
L-glutamine | Gibco | 25030-081 | 200mM |
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 | 0.1mm deep |
Trypan blue stain | Gibco | 15250-061 | |
Sterile 0.9% NaCl injection, USP | Hospira | NDC: 0409-4888 | 10ml vials |
0.6ml conical polypropylenemi microtubes | Genesee Scientific | 22-272 | |
Atipamezole hydrochloride injection | Orion Pharma | NDC: 52483-6298 | 5mg/ml solution |
Ketamine hydrochloride injection | Fort Dodge | NDC: 0409-2051 | 100mg/ml solution |
Dexmedetomidine hydrochloride injection | Zoetis | NDC: 54771-2805 | 0.5mg/ml solution |
Xylazine hydrochloride injection | Lloyd | NDC: 61311-481 | 100mg/ml solution |
Carprofen injection | Pfizer | NDC: 61106-8501 | 50mg/ml solution |
Buprenorphine hydrochloride injection | Reckitt Benckiser | NDC: 12496-0757 | 0.3mg/ml ampuls |
1ml tuberculin syringes | Covidien | 8881501400 | |
26G x ½ (0.45mm x 13mm) 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 |
1.7ml 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; 5μl volume |
Microliter syringe needles | Hamilton | 7762-06 | 33G, 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 | 7.9mm diameter x 50mm length (5/16" diameter x 2" length) |
1L glass screw-cap storage bottle | Corning | 1395 | Type 1, Class A borosilicate glass |
Heparin sodium | Sagent | NDC: 25021-400 | 1,000U/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 |
20G x 1 ½" aluminum hub blunt needles | Kendall | 8881202363 | 0.9mm x 38.1mm |
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. 115mm, base I.D. 85mm, 203ml 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 | |
7ml 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 |
70μm 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 | |
15ml polypropylene centrifuge tubes | Genesee Scientific | 21-103 | conical bottom |
50ml polypropylene centrifuge tubes | Genesee Scientific | 21-108 | conical bottom |
12x75mm 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 |