This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.
Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell's arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.
High resolution intravital optical imaging has proven to be crucial to understanding many biological processes, allowing single-cell and sub-cellular parameters to be measured and quantified. In cancer research, intravital imaging of tumor and stromal cells has led to the discovery of many microenvironmental interactions 6-11 that are only present in the intact animal.
Discoveries about microenvironments associated with intravasation and dissemination of tumor cells in breast cancer using single cell resolution optical imaging in vivo has even led to novel markers for prognosis and response to treatment in breast cancer patients 12-16. The best imaging technologies available for viewing deep within intact internal vital organs are the clinical modalities (MRI, PET, CT) which offer excellent views of the entire organ and can reveal pathologies even before they produce clinical symptoms. They are unable, however, to reveal the earliest stages of metastasis and the cellular mechanisms driving tumor progression due to their lack of single cell resolution. By the time lung metastases are visible in these modalities, they are well established and proliferating. Given the estimate that 90% of disseminated tumor cells that arrive to the lung either do not survive 17 or initially remain dormant 18, and the observation that they arrive much earlier than previously expected 19, imaging the earliest steps of arrival and survival becomes crucial to understanding the process of metastatic seeding and recurrence of tumor growth at distant sites.
Performing these observations in the lung has proven extremely difficult however; the vast majority of imaging studies have utilized ex vivo or explant preparations 20-23, which only give a view into the lung at single time points. While these preparations do provide useful information, they do not give a complete understanding of the interactions, cause and effect relationships, and dynamics that occur between the various components of the microenvironment. The lack of a proper circulatory system (and concomitant imbalance of homeostasis) and the disconnection from the rest of the body's immune system makes it desirous to validate the conclusions that these preparations generate in intact tissue in vivo.
Many groups have performed intravital imaging of the intact lung 2,4,5,24-33 with Wearn and German being the first to surgically expose the pleural layer 24 and Terry the first to utilize an implantable imaging window 25.
High resolution imaging in the lung is greatly hindered by the lung's constant motion and several techniques have been developed to overcome this limitation. Wagner and Filley 27 studied the natural motion of the canine lung and designed their surgical protocol to locate their implanted window over a relatively stationary region while Wagner utilized vacuum in his window surgical preparation to immobilize the tissue 28. Since that time, a variety of techniques have been utilized to image the lung including: bronchus clamping, sequential apnea and gated imaging, oversampled acquisition, gluing of the lung lobe and vacuum 34. Each of these has its advantages and disadvantages and no one technique has emerged as superior to another 34. For example, bronchus clamping and sequential apnea alter the normal exchange of gases in the lung and may cause atelectasis. Gated imaging and oversampled acquisition do not suffer from these disadvantages but require high-speed or specialized imaging equipment not widely accessible. Finally both gluing of the lung and the vacuum technique avoid both of the above mentioned drawbacks, but may exhibit shear force induced injury if care is not taken. In recent years, the vacuum window has been miniaturized and adapted for use in mice using confocal and multiphoton microscopy 4,5,33 and excellent high-resolution imaging has been attained 2. Table 1 summarizes this rich history and highlights those papers which describe novel advancements in the use of intravital lung imaging windows.
This protocol describes the use of extended time-lapse multiphoton intravital microscopy to image metastasis in the live, intact lung with the highest subcellular resolution possible. Images are acquired for up to 12 hr using a multiphoton microscope equipped with a high numerical aperture objective lens and multiple photomultiplier tube (PMT) detectors. Transgenic mouse models are utilized to fluorescently label native macrophages along with fluorescent high molecular weight dextran and fluorescent protein transfected tumor cells (to label the vasculature and tumor cells respectively). While this choice of fluorescently labeled cells enables visualization of tumor cell-endothelial cell-macrophage interactions and dynamics, this protocol will work for any strain of fluorescent or non-fluorescent mouse. After acquisition, residual drift motion (if any) is eliminated using a Fiji plugin 35,36 and custom macros time average the vascular channel to eliminate flashing caused by unlabeled circulating blood cells.
While this protocol focuses on imaging metastasis, the techniques are applicable to many other biological processes observable with high-resolution single-cell imaging in the lung.
All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.
1. Generating Fluorescently Labeled Mouse Model and Tumor Cells
2. Multiphoton Microscope Set up and Imaging Preparation
NOTE: While this protocol can be performed on any multiphoton microscope, the system used to acquire the data shown in this protocol has been described previously in detail 45.
3. Vacuum System Set up
4. Surgery
5. Intravital Imaging
6. Euthanasia
7. Image Analysis
To demonstrate the type of results that can be achieved with this method, we injected E0771-LG tumor cells labeled with the fluorescent protein Clover into the tail vein of MacBlue mice 44 at varying time points before surgery. After surgery, 155 kD rhodamine labeled dextran was injected IV to mark the vasculature and time-lapse imaging was performed.
When imaging mice 24 hr post injection, single cells are visible inside of the vasculature, interacting with macrophages and monocytes. An example of this is shown in Figure 2A (Supplemental Movie 1). Here a single optical section of a solitary tumor cell (green) 12 µm deep lodged in the lung vasculature is imaged over 5 hr and 20 min as it transiently interacts with a resident macrophage (cyan). Vasculature is labeled by high molecular weight dextran. The stability of the imaging is such that sequential z-stack imaging of the field can be acquired and a 3 dimensional reconstruction can be made (Figure 2B, Supplemental Movie 2).
Use of the microscope settings described in the protocol for mosaic imaging allows imaging of structures larger than a single field of view. For example, Figure 3 demonstrates acquisition of a single metastatic lesion, 12 days post injection. This 5 x 5 mosaic shows an 890 µm field of view over 205 min at 15 (Figure 3A, left panel; Supplemental Movie 3) and 25 µm (Figure 3A, right panel) below the surface of the lung. Despite the large field of view, the high resolution of the underlying frames composing the mosaic enables the capture of subcellular events such as mitosis of a single cell as evidenced by chromosomal separation (Figure 3B, Supplemental Movie 4).
Intravascular injection of a high molecular weight fluorescently labelled dextran results in labeling of the vascular lumen, however unlabeled circulating erythrocytes and leukocytes occlude the dextran. In the small capillaries of the lung, the occlusion is complete resulting in a flashing of the dextran signal and a loss of definition of the vasculature boundaries (Figure 4, left; Supplemental Movie 5). The high spatial stability provided by this protocol allows time averaging of the blood channel, without blurring, thus restoring the temporary occlusions. The other signal channels can then be overlaid on the vasculature to provide a clear view of the vessel boundaries (Figure 4, right; Supplemental Movie 6).
Figure 1: Layout of the Vacuum System. House vacuum is utilized and set to defined level with a vacuum regulator. A capture flask prevents contamination of the regulator and vacuum system by bodily fluids. A thin flexible tube conveys the vacuum to a pipette tip cut to fit into the vacuum port of the imaging window. The imaging window is fit into a recessed groove in the imaging plate maintaining its positional stability with respect to the microscope objective lens. Please click here to view a larger version of this figure.
Figure 2: Single Cell Imaging in the Lung. (A) Still from a time lapse movie of a single tumor cell in the capillary bed of the lung 24 hr after tail vein injection. (Red = Blood vessels, Green = Tumor Cells, Cyan = Macrophages) (B) Stable imaging allows three dimension reconstruction of imaging data over time. Blood vessels have been time averaged for clarity. (Red = Blood vessels, Green = Tumor Cells, Blue = Macrophages). Please click here to view a larger version of this figure.
Figure 3: The Stability of the Lung Allows High-resolution, Large-field of View Imaging in the Lung by the Sequential Acquisition and Stitching Together of Multiple Low Magnification Fields. (A) 5 x 5 mosaic showing an 890 µm field of view of a metastatic lesion in the lung 12 days after tail vein injection of tumor cells taken at 15 µm below the lung surface (Left Panel). Right panel shows the same metastatic lesion at 25 µm below the lung surface. (B) The individual high resolution fields reveal subcellular processes such as chromosomal alignment (yellow arrows) and separation (red arrows) during cell division. Please click here to view a larger version of this figure.
Figure 4: Intravascular Injection of Fluorescently Labeled High Molecular Weight Dextran Marks the Lumen of the Vasculature Except when Unlabeled Erythrocytes and other Circulating Cells Occlude the Fluorescence Signal. (A) Occlusion results in an incomplete labeling which moves in time resulting in a flashing effect obscuring the vessel boundaries. (B) The high spatial stability of the vacuum window allows the blood channel to be time averaged, filling in the temporary occlusions and clearly defining the vessel boundaries. The other channels are then overlaid without averaging. Please click here to view a larger version of this figure.
Year | First Author / Last Author | Animal | Surgery | Type of Imaging | Notes |
1926 | Wearn & German | Cats | Chest wall cut down to pleural layer. Second opening made through diaphragm down to pleura for illumination. | Bright-field microscopy. | First “window” through pleural wall. |
1939 | Terry | Cats | Ribs are separated, 1 in. window implanted, air removed from thorax with vacuum. | Binocular and skin microscope using polarized illumination. | First implanted optical window. Used vacuum to draw tissue to window. |
1963 | de Alva & Rainer | Rabbits & Dogs | One or two ribs are resected and 1 in. window inserted and sutured to chest wall. Skin was closed over window and animal allowed to heal. Before imaging, skin was dissected to expose window. | Used high speed stroboscopic cinematography through a low mag (11X) objective. | First survival window. |
1965 | Wagner & Filley | Dogs | Right forelimb removed, one rib cut and window inserted and sutured. | Reflection microscopy with 22X objective. | First relatively motion free window without vacuum. |
1969 | Wagner | Dogs | One Rib is resected, 3 in. window implanted. Threaded flange screws on window to form seal to chest wall. | Brightfield microscopy with high-magnification 100X oil immersion objective. | First window to use vacuum to stabilize tissue movement. |
1992 | Groh & Goetz | Rabbits | One rib resected, 1.2 in. window implanted. Threaded flange screws on window to form seal to chest wall. Vacuum applied. | Epifluorescent microscopy with 25X objective. | First use of a vacuum window with epifluorescence. |
1994 | Fingar & Wieman | Rats | 2 ribs are resected, 1 in. window implanted and sutured to chest wall and skin. | Epifluorescent microscopy with 40X objective. | First survival window in rats. |
2000 | Funakoshi & Mitsui | Mice | Entire chest wall removed. Vacuum suction ring attached to right lung. | Confocal microscopy with a 20X objective. | First use of vacuum window in mice. |
2005 | Lamm & Glenny | Rats | Entire chest wall removed, 1 in. window attached to lung. | Reflection and epifluorescent microscopy with a 20X objective. | First use of vacuum window in rats. |
2008 | Tabuchi & Keubler | Mice | 3 ribs are resected, plastic film applied to opening to seal hole in chest wall. Air removed via intrapleural catheter. | Epifluorescent microscopy with 20X objective. | First to use plastic film to seal opening in chest wall. |
Table 1: Historical Survey of the Development of Intravital Lung Imaging Windows. Many novel intravital lung imaging windows have been developed over the years with the most recent being miniaturized for use in mice, employing vacuum for tissue stabilization and attaining high enough resolution to be capable of revealing subcellular detail.
Supplemental Figure 1: Design Drawing of the Vacuum Imaging Window Please click here to download this figure.
Supplemental Figure 2: Photographs of Vacuum Setup Please click here to download this figure.
Supplemental Figure 3: Design Drawing of Stage Plate Insert Please click here to download this figure.
Supplemental Movie 1: Movie of stills in Figure 2A. (Right click to download).
Supplemental code file: Mosaic Stitching. Please click here to download this file.
Supplemental code file: Perform Blood Averaging. Please click here to download this file.
High resolution in vivo optical imaging combined with fluorescently labelled functional tags such as proteins and antibodies has dramatically increased our understanding of the metastatic cascade. It has enabled direct visualization and quantification of single-cell and sub-cellular parameters in tumor cells, host cells and their microenvironment. This imaging within the primary tumor has led, for example, to the discovery of discrete microenvironments that are supportive of either growth invasion or dissemination 6,7. In the case of invasion, in vivo imaging has revealed the preferential role of co-migrational streaming of macrophages and tumor cells in intravasation 7,50.
In secondary sites like the lung, understanding the dynamics of tumor cell behavior at the earliest stages of metastasis, including the pre-micrometastasis seeding stage when single and small groups of tumor cells arrive and interact with the blood vessel endothelium, will only be accomplished using high resolution optical imaging. Standard clinical imaging modalities do not have the resolution needed to visualize either the fine structure of the capillary bed or the morphology and interaction of cells at single cell resolution. The imaging techniques presented in this protocol accomplish this challenging task.
Intravital imaging windows such as those listed in Table 1 offer a significant advantage over ex vivo lung preparations by maintaining proper lung physiology including perfusion, connection to the immune system and offering more than just a static view of cellular dynamics. The vacuum stabilized windows in particular offer a level of tissue stability that allows the acquisition of highly registered images, enabling three dimensional reconstructions (Figure 2B) and the capability for large field of view mosaic imaging (Figure 3A). Together these provide a range of views of the lung, from a histologic type low magnification view which gives the tissue morphology, to a sub cellular view that can even reveal chromosomal separation and discriminate dormant and dividing tumor cells (Figure 3B). Multiple acquisition channels allow several cell types and their interactions to be visualized simultaneously (Figure 2A).
While the protocol does take some technical skill, practice and attention to some critical steps and points will improve the success rate of the procedure and imaging times of up to 12 hr can be expected. It is critical to make sure the lung tissue is well centered over the window (step 4.25). This ensures that the vacuum is applied evenly and completely across the lung tissue (step 4.27). Failure to center the tissue will result in motion of the tissue. The restraining harness (Supplemental Figure 2) is used to reduce the motion induced by constriction of the intercostal muscles during the natural breaths the animal takes. It should be snug over the mouse, but not compress the chest. Too much compression puts pressure on all of the lobes of the lungs as well as the heart and results in a reduced viability of the mouse. If dextran is not observed flowing in the lung vasculature after injection (step 5.8), the lung tissue is either damaged from improper handling during surgery or the vacuum level is too high. Lowering the vacuum by .5-1 inch Hg can be attempted to see if flow is restored. If the flow is not restored, or if there is flow, but dextran is observed extravascularly, the lung tissue has been damaged in that region and it will be necessary to image a different field of view. Maintenance of proper blood flow in the imaging region is important to ensuring that proper physiology is being measured. Since oxygen is supplied to the tissue from inside of the alveoli and not through the vasculature, ischemic hypoxia is unlikely. Still, unperfused vasculature can potentially lead to altered oxygen/CO2 levels and will also prevent circulating leukocytes from reaching the tissue of interest. Visualization of flowing erythrocytes can be used as an indicator of the proper lung function. Lung tissue is very delicate and also susceptible to photo-damage. If after time lapse imaging blood flow stops in the imaged field, the laser is most likely too high and subsequent imaging of other fields must be done at lower power. We have found ~10-15 mW at the sample to produce sufficiently bright images without photodamage when using either GFP or Clover (which has four times the mean brightness) transfected cells. Minimum brightness levels for the fluorescent proteins are highly dependent upon microscope parameters and the expression levels in the cells and must be tested empirically.
In this protocol, tumor cells are labeled with a bright cytoplasmic fluorescent protein that offers a clear view of the cell body and intracellular spaces that exclude the protein (i.e. nucleus). Macrophages are labeled by utilizing a transgenic mouse model syngeneic to the tumor cells. Labeling both cell types enables visualization of their direct interaction in real time. The extended duration of imaging allows quantification of the frequency, duration and extent with which cancer cells interact with macrophages in a physiologically relevant context.
When performed correctly, this procedure enables motion free, multiple-channel, high-resolution, single-cell imaging in the intact lung for periods of up to 12 hr. The use of a high numerical aperture objective lens such as the 25X 0.95NA and the multiphoton's capabilities for electronic zoom allows the highest resolution optical imaging in the lung seen to date.
Intravascularly injected fluorescent, high molecular weight dextran performs the dual role of labeling the vascular space and verifying its integrity. 155 kDa dextran is used to prevent diffusion through the interendothelial spaces. Any sign of a lack of vascular flow or of vascular leakage into the extravascular space indicates damage to the tissue due to improper handling or excessive vacuum.
Finally, unique image processing techniques can be employed that take advantage of the high spatial stability of this protocol. Since the unlabeled erythrocytes and other leukocytes exclude the fluorescent dextran when they pass through the capillaries, this signal can be averaged over time to remove the flashing that they create. This offers a well-defined view of the vasculature not possible otherwise.
Limitations of this technique include both the invasive nature of the surgery, which potentially complicates its use for study of diseases which weaken the animal (e.g. late stage metastatic cancer, acute sickle cell anemia), and the fact that the surgery is terminal which limits it use to a single, albeit long (up to 12 hr), imaging session. Further, given the long duration of the imaging session and the low hepatic glycogen reserve of mice 51, glucose supplementation may be given to avoid potential sources of bias in experiments.
This protocol could potentially be modified with injectable fluorescently labeled antibodies either in place of or in addition to the dextran in order to label other structures or cell types in real time. This will expand the capabilities for analyzing and dissecting the tumor microenvironment by directly visualizing tumor cell-host cell interactions and dynamics in real time.
The authors have nothing to disclose.
This research was supported by NIH-CA100324, Einstein National Cancer Institute’s cancer center support grant P30CA013330, R01CA172451 to JWP and the Integrated Imaging Program. This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at the Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work. The authors thank Mike Rottenkolber, Ricardo Ibagon and Anthony Leggiadro of the Einstein machine shop for their skilled and timely craftsmanship, the laboratory of Matthew Krummel for generously sharing their window design drawings, Kevin Elicieri and Jeremy Bredfeldt for their expertise in microscopy and their amplifier recommendations and Allison Harney and Bojana Gligorijevic for informative discussions.
Nickel-Plated Brass Vacuum Regulator 1/8 NPT Female, w/ Gauge, 0 – 20" Hg Vacuum | McMaster Carr | 4172K12 | Vacuum Regulator |
Brass Barbed Hose Fitting Adapter for 1/4" Hose ID X 1/8" NPTF Male Pipe | McMaster Carr | 5346K13 | Vacuum Regulator Hose Adapter |
Pyrex Brand Filtering Flasks with Tubulation; Neck tooled for rubber stopper No. 4; Capacity: 50mL | Corning Life Sciences Glass | 5360-50 | Vacuum Flask |
Round Glass Coverslips Thickness #1.5, 0.16-0.19mm 10mm dia. | Ted Pella, Inc. | 260368 | Cover slips |
Exel International Disposable Safelet I.V. Catheters; 22gx1 in. | Exel International | 26746 | Tracheal Catheter |
PERMA-HAND Black Braided Silk Sutures, ETHICON LIGAPAK Dispensing Reel Size 2-0 | VWR | 95056-992 | String |
Liquid Super Glue, Clear, 0.14oz | Hendel Corp. | LOC1647358 | Cyano-acrylate Glue |
Tetramethylrhodamine isothiocyanate–Dextran | Sigma-Aldrich | T1287-500MG | 155kD Dextran |
Laboratory Clear Tygon PVC Tubing, 1/16" ID, 1/8" OD, 1/32" Wall Thickness, 25 ft. Length | McMaster Carr | 5155T12 | Thin Tubing & Tubing for Luer |
Crack-Resistant Polyethylene Tubing, 1/8" ID, 1/4" OD, 1/16" Wall Thickness, White, 50 ft. Length | McMaster Carr | 5181K24 | Thick Tubing |
Depillatory Lotion | Nair | – | |
Micro Medical Tubing 95 Durometer LDPE | Scientific Commodities Inc. | BB31695-PE/1 | Tubing for tail vein catheter |
30 G x 1 in. BD PrecisionGlide Needle | BD | 305128 | Needles for tail vein catheter |
Puritan Nonsterile Cotton-Tipped Swabs | Fisher Scientific | 867WCNOGLUE | |
Clear Polycarbonate Barbed Tube Fitting, Reducing Straight for 3/32" x 1/16" Tube ID | McMaster Carr | 5117k51 | Connectors between tubes |
One-Hole Rubber Stoppers | Fisher Scientific | 14-135F | Stopper for Vacuum Flask |
SHARP Precision Barrier Tips, For P-100, 100µl | Denville Scientific Inc. | P1125 | Pipette Tip |
Laboratory tape | Fisher Scientific | 159015R | |
Puralube | Henry Schein Animal Health | 008897 | Opthalmic Ointment |
Gemini Cautery Kit | Harvard Apparatus | 726067 | Cautery Pen |
Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4" Length | Roboz Surgical | RS-5135 | Forceps |
Extra Fine Micro Dissecting Scissors 4" Straight Sharp/Sharp 24mm | Roboz Surgical | RS-5912 | Sharp Scissors |
Micro Dissecting Scissors 4" Straight Blunt/Blunt | Roboz Surgical | RS-5980 | Blunt Scissors |
Wipes | Fisher Scientific | 06-666-A | Harness |
PhysioSuite System | Kent Scientific | PhysioSuite | Vitals Monitor |
1 mL Syringe, Tuberculin Slip Tip | BD | 309659 | Syringe |
Cyano acrylate | Staples | LOC1647358 | Cover Slip Adhesive |
Petroleum Jelly | Fisher Scientific | 19-086291 | Water Barrier |
Adapter Luer Cannulla 1.5-2.2mm | Harvard Apparatus | 734118 | Catheter Connector |
MouseOx oximeter, software and sensors | STARR Life Sciences | MouseOx | Pulse Oximeter |
Isoethesia (isoflurane) | Henry Schein Animal Health | 50033 | 250 mL |
Oxygen | TechAir | OX TM | |
1 x PBS | Life Technologies | 10010-023 | |
PVC Ball Valve, Push to Connect, 1/4 In | Grainger | 3CGJ7 | Vacuum Valve |
Small Animal Ventilator | Harvard Apparatus | 683 | Alternative is available from Kent Scientific: MouseVent |
OptiMEM Reduced Serum Medium | ThermoFisher Scientific | 31985062 | |
Lipofectamine 2000 Transfection Reagent | ThermoFisher Scientific | 11668019 | |
MacBlue Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume/J Mice | Jackson Laboratory | 026051 | |
Multiphoton Microscope | Olympus | Fluoview FV1000 | Alternative to custom built scope |
Environmental Enclosure | Precision Plastics | Chamber for FV1000 | Alternative to custom built enclosure |
Phosphate Buffered Saline | ThermoFisher Scientific | 14190136 | |
Laser Power Meter | Coherent | FieldMaxIITOP | |
Laser Power Meter Head | Coherent | PM10 | |
pcDNA3-Clover Fluorescent Protein Vector | Addgene | 40259 | |
G418 Sulfate Selective Antibiotic | ThermoFisher Scientific | 10131027 | |
MoFlo Fluorescent-Activate Cell Sorter | Beckman Coulter | XDP | |
Trypsin EDTA 1X | Corning | 25-052-Cl | |
40 µm Mesh | Falcon | 352235 | |
96 Well Plate | Costar | 3599 | |
60 mm Culture Dish | Corning | 430196 | |
10 cm Culture Dish | Corning | 353003 | |
Bovine Serum Albumin | Sigma-Aldrich | A4503 | |
Dulbecco's Phosphate Buffered Saline 1X | Corning | 21-031-CV | |
C57BL/6J Mouse | Jackson Laboratory | 000664 | |
Kim Wipes | Fisher Scientific | 06-666-A |