This work was financially funded by Geran Putra Berimpak (GBP), Universiti Putra Malaysia: 9542800. We would like to thank the Agro-Biotechnology Institute, Malaysia (ABI) laboratories, and microscopy imaging facility. Special thanks to Mohd Daniel Hazim for help with editing and recording the experimental video for this work.

NOTE: Sections 1&#8722;6 describe the coculture model of 4T1 mouse mammary carcinoma cells and RAW 264.7 mouse macrophages. Section 7 describes the time-lapse assessment of M1 macrophages phagocyted 4T1 cells.
1. Culturing 4T1 mouse mammary carcinoma cells and RAW 264.7 mouse macrophages
<ol>
	<li>Thaw a vial of cryogenically preserved 4T1 and RAW 264.7 passage 6 cells by gentle agitation in a water bath at 37 &#176;C or the normal growth temperature for the cell lines. 
	NOTE: Thawing should be done rapidly, approximately within 2 min. To avoid contamination, remove the vial from the water bath and decontaminate it by spraying with 70% ethanol. To avoid genetic drift, especially for the macrophages, use a low passage number (i.e., less than 20).</li>
	<li>Dilute the thawed cells in 10 mL of complete DMEM medium in a 15 mL conical tube and centrifuge the cells at 600 x g for 5 min to obtain a cell pellet. 
	NOTE: Complete DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS), 200 U/mL penicillin/streptomycin, and 2 mM L-glutamine is used throughout the protocol.</li>
	<li>Carefully aspirate the media, resuspend the cells in 8 mL of complete medium and transfer into a 25 cm2 tissue culture treated flask. Culture both 4T1 and RAW 264.7 cells in complete DMEM medium at 37 &#176;C with 5% CO2. 
	NOTE: Resuspend the cell pellets gently and add the cell suspension into the culture flask drop by drop to avoid killing the cells.</li>
	<li>After 1 week of culturing to allow cell adherence, monitor the flask daily and add 8 mL of complete DMEM medium as needed.</li>
</ol>
2. Immunostaining of M1 polarized RAW 264.7 macrophages
<ol>
	<li>As the confluency of RAW 264.7 cells reaches 70&#8722;80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS. 
	NOTE: Before seeding RAW 264.7 cells, make sure no cell differentiation has occurred (i.e., dendrite-like phenotype and/or increased cell size). If cell morphology changes are visible, discard the culture and thaw a new cell line from the earlier passage vial.</li>
	<li>Prepare the macrophages for collection by gently dislodging the adherent cells using a cell scraper and transfer them into a 15 mL conical tube.</li>
	<li>Count the cells using a hemocytometer and prepare a cell suspension of 1 x 105 cells/dish using complete DMEM medium.</li>
	<li>Seed 2 mL of the macrophages suspension into two imaging dishes. Incubate the cells 2&#8722;3 h at 37 &#176;C with 5% CO2 to allow cell adherence.</li>
	<li>Prepare M1 polarization media by adding 100 ng/mL LPS and 20 ng/mL IFN-&#947; into complete DMEM medium10,11.</li>
	<li>Discard the culture supernatant from the macrophages and carefully wash the monolayers in the dish 2x with at least 1 mL of PBS.</li>
	<li>Add 2 mL of M1 polarization media into one of the imaging dishes, allow the RAW 264.7 cells to induce the M1 macrophage phenotype, and incubate 2&#8211;3 h at 37 &#176;C with 5% CO2. 
	NOTE: Seed RAW 264.7 macrophages in an imaging dish with DMEM supplemented with 10% FBS as a control for the anti-iNOS antibody staining. Label the imaging dishes RAW (control) and M1 macrophages (LPS and IFN-&#947; polarized), respectively.</li>
	<li>Before immunostaining, fix the RAW 264.7 and M1 macrophages cells using 2 mL of 4% paraformaldehyde and incubate for 15 min at 37 &#176;C (room temperature, RT).</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Quench with 2 mL of 50 mM ammonium chloride in PBS and incubate for 15 min at RT.</li>
	<li>Permeabilize using 2 mL of 0.3% Triton X-100 in PBS for 15 min at RT.</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Block using 2 mL of 10% FBS in PBS for 30 min at RT. 
	NOTE: The blocking step in immunostaining is to minimize the non-specific binding of antibody within the cell.</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Incubate with 2 mL of anti-iNOS-FITC in PBS and 1% FBS overnight at 4 &#176;C. 
	NOTE: Label the cells using the appropriate antibody dilution according to the manufacturer's recommendations. Protect the dishes from light by covering them with aluminum foil from this point on.</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Incubate 1 mL of 300 nM 6-diamidino-2-phenylindole (DAPI) into imaging dishes for 10 min in 37 &#176;C, in the dark by covering with aluminum foil.</li>
	<li>Wash cells with 1 mL of PBS at least 3x and add 1 mL of complete DMEM medium into the imaging dishes. 
	NOTE: The cells are now ready for fluorescence imaging. The microscope setup will need an inverted stage and excitation filters for the chosen stains (in this case, FITC and DAPI).</li>
	<li>Turn on the microscope and load the imaging software. Mount the imaging dish on the microscope and adjust the focus to observe RAW 264.7 and M1 macrophages. Optimize the appearance of phase-contrast FITC and DAPI images by adjusting the transmitted light and exposure times. 
	NOTE: Begin imaging when all points are in focus and the channels are optimized.</li>
	<li>Capture phase-contrast FITC and DAPI images (Figure 1).</li>
</ol>
3. Seeding 4T1 mouse mammary carcinoma cells
<ol>
	<li>As the confluency of 4T1 cells reaches 70&#8722;80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS. Add 1 mL of prewarmed trypsin to dissociate the cells and incubate for up to 20 min at 37 &#176;C. 
	NOTE: Observe the cells under a microscope. Detached cells will be rounded.</li>
	<li>Once the cells detach, transfer them to a 15 mL conical tube and add 2 mL of prewarmed complete DMEM medium to inactivate the trypsin. Gently disperse the medium by pipetting the cell layer surface to recover the cells. Then, centrifuge at 600 x g for 5 min to obtain a cell pellet.</li>
	<li>Remove the supernatant and resuspend the pellet in 10 mL of prewarmed complete DMEM medium.</li>
	<li>Count the cells using a hemocytometer and seed 1 x 105 4T1 cells in 2 mL of complete DMEM medium in each of two 35 mm glass-bottom imaging dishes treated with tissue culture. Incubate the cells overnight at 37 &#176;C with 5% CO2. 
	NOTE: Culture 4T1 cells in one of the imaging dishes with M1 polarization media and label 4T1-Inducer (control). This is to ensure normal growth of 4T1 cells when exposed to the inducers.</li>
</ol>
4. Labeling living 4T1 mouse mammary carcinoma cells using CFSE staining
<ol>
	<li>First, remove the existing media from the 4T1 cells imaging dishes. Wash the cell monolayer at least 2x with 1 mL of PBS.</li>
	<li>Prepare 5 &#181;M of CFSE staining solution by diluting CFSE in 1 mL of PBS. Add 1 mL of 5 &#181;M CFSE staining solution into each imaging dish and incubate the cells for 20 min at RT or 37 &#176;C, in the dark. 
	NOTE: Label the cells using appropriate CFSE working concentration according to the manufacturer's recommendations and preliminary experiments to obtain the optimum CFSE staining concentration. Labeling efficiency will be higher if CFSE is diluted in PBS.</li>
	<li>Quench the staining by adding an equal volume of complete DMEM medium containing 10% FBS and staining solution to the cells and incubate for 5 min at RT in the dark.</li>
	<li>Discard the CFSE-containing solution and wash the cells 1x with an equal volume of culture media. 
	NOTE: 4T1 cells are now fluorescently labeled.</li>
	<li>Return the 4T1-CFSE cells to standard culture conditions at RT with 5% CO2.</li>
</ol>
5. Seeding RAW 264.7 mouse macrophages and coculture with 4T1
<ol>
	<li>As confluency of RAW 264.7 reach 70&#8722;80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS.</li>
	<li>Prepare the macrophages for collection by gently dislodging the adherent cells using a cell scraper and transfer them into a 15 mL conical tube.</li>
	<li>Count the cells using a hemocytometer and prepare a cell suspension of 1 x 105 cells/mL by diluting the remaining solution with a complete DMEM medium according to the seeding density. 
	NOTE: Overly confluent cells in cocultures may severely reduce phagocytosis. In this study, the seeding ratio of macrophages: cancer cells was adjusted to a 1:1 ratio. The ratio can be adjusted depending on the aggressiveness of the tumor cells and the origin of the macrophages.</li>
	<li>Before coculturing, discard the culture supernatant from the 4T1 cells seeded a day before (step 4.5) and carefully wash the monolayers 2x with at least 1 mL of PBS.</li>
	<li>Seed 1 x 105 RAW 264.7 cells per 2 mL of complete DMEM medium into one of the 4T1 imaging dishes. Incubate the cells 2&#8722;3 h at 37 &#176;C with 5% CO2 to allow cell adherence. Label the imaging dish 4T1-M1 coculture. 
	NOTE: The 4T1 (control) cells were not cocultured with macrophages.</li>
</ol>
6. M1 polarization of RAW 264.7 macrophages
<ol>
	<li>Prepare M1 polarization media by adding 100 ng/mL LPS and 20 ng/mL IFN-&#947; into complete DMEM medium supplemented with 10% (v/v) FBS10,11.</li>
	<li>Discard the culture supernatant from the macrophages incubated before (step 5.5) and carefully wash the monolayers in the dish 2x with at least 1 mL of PBS.</li>
	<li>Then, add 2 mL of M1 polarization media into the imaging dish and allow the RAW 264.7 cells to induce into the M1 macrophage phenotype by incubating at 37 &#176;C with 5% CO2. 
	NOTE: M1 macrophage cells may take up 2&#8722;3 h of incubation to achieve complete polarization. Preliminary experiments need to be done to ensure the suitable incubation period to allow complete polarization of macrophages.</li>
</ol>
7. Live-cell video microscopy of phagocytosis
NOTE: Many factors need to be considered when performing live-cell imaging to obtain a producible video, including optimization of image exposure, time measurement, and automatic focus correction.
<ol>
	<li>Before the experiment, set up the cell culture incubator by turning on thermostat at 37 &#176;C and supplying 5% CO2. 
	NOTE: The microscope setup requires an inverted stage, a stage top incubator, humidity control (95%), and gas incubation system. This setup is crucial to maintain the cells for long-term live-cell video microscopy assessment.</li>
	<li>Turn on the microscope including the piezo stage and load the microscope imaging software.</li>
	<li>Position the seeded coculture cells in a 35 mm glass-bottom imaging dish in the center of the stage and gently screw on the top chamber. Use the joystick to motorize the stage by selecting the position to be imaged.</li>
	<li>To view the sample, select the phase-contrast filter on the turret. After initially viewing the sample, locate the appropriate fields and bring the sample into focus. 
	NOTE: Imaging software allows use of fully automated objective selection, Perfect Focus System, time-lapse series, multichannel image acquisition, and multipoint image acquisition.</li>
	<li>To set up multichannel fluorescence for CFSE labeling and differential interference contrast acquisition, open the imaging software acquisition dialog box and select the [Lambda] tab checkbox (Figure 2). 
	NOTE: For a 13 h time-lapse imaging, a phase-contrast channel should be used.</li>
	<li>Select [Lambda] tab | [Optical Configuration] and select [10X DIC] for differential interference contrast and [GFP-R] for the green fluorescence filter checkbox. Under the [Lambda] | [Focus] column, select the [10X DIC] checkbox to set as the focus reference.</li>
	<li>Open the imaging software acquisition dialog box and click the [XYZ Time &#8230;] button and select the [XY] tab.</li>
	<li>Move to the image acquisition point using the joystick on the motorized stage while checking the live image or select a different position through the eyepieces. Then click on the checkbox under the [Point Name] column to set each point in each location for image capture (see Figure 3). 
	NOTE: The automated stage allows multipoint image acquisition of different XY coordinates to capture multiple fields.</li>
	<li>Fine-tune the focus for each sample viewed on the screen. Use the controls on Auto-focus correction.</li>
	<li>Use the software to set up time-lapse image capture. In the imaging software acquisition dialog box, check the [Time] tab (see Figure 4).</li>
	<li>Determine the [Interval] (the delay between the beginning of one time point to another time point) and [Duration] (the total length of time of the experiment). Units of time vary and can be selected in milliseconds (ms), seconds (s), minutes (m), or hours (h) from the dropdown menu. 
	NOTE: The imaging period length for time-lapse imaging of fluorescence and DIC time-lapse multichannel acquisition may vary depending on the fluorescent dye used in this assay. Photobleaching may occur if the labeled cells are exposed for too long.</li>
	<li>Check the [Save to File] checkbox to save acquired images. Under the [Time schedule] tab click the [Run now] button to acquire multipoint time-series images (see Figure 5). 
	NOTE: In the imaging software, images are saved in nd2 file format. Each time-lapse can be individually saved in an MP4 file format later.</li>
</ol>

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The authors have nothing to disclose.

The protocol described requires two steps: 1) coculture of 4T1 mouse mammary carcinoma cells and M1-polarized macrophages, and 2) assessment of the macrophage phagocytic activity using time-lapse microscopy. Live cell coculture is widely used in phagocytosis and migration assays. The live cell coculture model here is a simple, adaptable in vitro procedure (Figure 2) that utilizes a fluorescent dye, CFSE, which is used to label the 4T1 targeted cells. This is used for proper tracking of the cell types during live-cell imaging. Time-lapse live-cell imaging was used to assess the phagocytic activity of M1 macrophages using multipoint time-lapse 2D imaging before the 13 h of coculture.
The live cell coculture model was established using 4T1 mouse mammary carcinoma cells and M1 mouse macrophages. To distinguish the cells from one another, 4T1 cells were stained using a CFSE fluorescent dye (see section 4). CFSE allows for tracking the cells and monitoring cell division. CFSE can passively diffuse into cells, making CFSE staining a suitable cell tracker to visualize phagocytosis of targeted cells. As phagocytic macrophages digest and engulf cancer cells, they take up the CFSE dye and eventually become fluorescent12,13. It is essential to seed 4T1 cells in the imaging dish before the M1 macrophages. This is due to the different sizes and shapes of both cells. The 4T1 cells have an epithelial morphology that proliferates in small clusters. Also, the 4T1 cells must be stained with CFSE before coculture with the unstained M1 macrophages. Before macrophage polarization using LPS and IFN-&#947;, the murine macrophages RAW 264.7 are round, small, and usually grow as single cells. After LPS and IFN-&#947; polarization, induced M1 macrophages have a relatively flattened, pancake-like shape, with a porous cytoplasm14,15. To ensure proper polarization of RAW 264.7 towards M1 state, 100 ng/mL LPS and 20 ng/mL IFN-&#947; stimulated macrophages were immunostained with an M1 marker, anti-iNOS antibody conjugated to FITC (Figure 1). This protocol is performed before coculturing the macrophages and targeted cells to ensure that the macrophages are completely polarized into the M1 state.
This study describes a method for fluorescent microscopy to visualize the phagocytic activity of living macrophages. To minimize photobleaching and to provide better fluorescence imaging, fluorescent microscopy can be combined with other imaging techniques that are nondestructive to the fluorochrome, similar to DIC. The present protocol describes the materials and methods necessary for the assessment of the phagocytosis of mouse mammary carcinoma cells by LPS and IFN-&#947; stimulated macrophages using fluorescent and DIC time-lapse microscopy. Nonetheless, fluorescent microscopy studies have limitations when studying live-cell imaging compared to phase-contrast time-lapse imaging. During fluorescent live-cell imaging, prolonged exposure to a high amount of excitation light can induce serious cell damage16. Photobleaching can cause significant problems in live-cell imaging. The high-intensity illumination used in live-cell imaging can reduce the ability of CFSE dye to fluoresce. Nevertheless, a 13 h phagocytosis assessment was achieved in the second part of this study using phase-contrast time-lapse 2D imaging (Movies 2&#8211;5).
Time-lapse microscopy offers advantages such as generation of data from a single experiment at any time point throughout the desired duration period and significantly, multiple stage points within a single imaging dish can be captured for a single experiment. This allows observation of various positions in a single experiment. The protocol can also be used using multiple wells from culture plates (e.g., 6, 24, 96 well plates). Among the most critical technical challenges to perform successful live-cell imaging experiment includes maintaining the cells in a healthy condition by using a stage top incubator to provide a stable temperature of 37 &#176;C, 95% of humidity, and 5% CO2. Because the experiment took 13 h, ensuring that the microscope functions normally throughout was crucial.
During this study, six different points in the imaging dish were captured throughout a 13 h duration for 15 min intervals per point (Movie 2). The points can be adjusted depending on the event of the cell under investigation. Considering there are multiple stage points to be captured throughout this experiment, it is significant to make sure that each point has a stable focus before performing the live-cell video recording. The time interval for investigation must be optimized. A decreased time interval will have more detailed points that can be imaged; however, the file size will be very large. Increasing time interval will result in a lengthy video and will make the movie lose continuity.

Macrophages are the first line of immune defense and play a role in orchestrating immune responses against pathogens and foreign materials, including cancer cells. They are a specialized phagocyte that destroys and gets rid of unwanted particles in the body. Macrophages contribute defensive functions such as the clearance of apoptotic cells and microorganisms and the recruitment of other immune cells1. Macrophages can differentiate into two different types, M1 and M2 macrophages, in response to environmental signals2. M1-polarized macrophages (i.e., classically activated macrophages) are activated by the cytokine interferon-&#947; (IFN-&#947;) and lipopolysaccharides (LPS) and are involved in the inflammatory response, pathogen clearance, efficient phagocytosis, and tumoricidal immunity3,4. The M2 macrophages are closely related to tumor-associated macrophages (TAMs) and have anti-inflammatory and tumor promotion properties4.
Phagocytosis, derived from Ancient Greek (phagein), meaning "to devour," (kytos), meaning "cell," and -osis, meaning &#34;process&#34;5. Phagocytosis is a receptor-mediated process where phagocytes (including macrophages, monocytes, and neutrophils) kill and engulf invading pathogens, clean up foreign particles, and clear apoptotic cell debris. Tumor-associated macrophages (TAMs) are found in the stroma in different tumors, including breast cancer, and have pro-tumor functions6,7, resulting in resistance to phagocytosis. The detailed mechanism of tumor cell phagocytosis by macrophages is not yet understood.
This study presents a two-step method: 1) 4T1 mouse mammary carcinoma cells and M1-polarized macrophages are cocultured, and 2) the phagocytic activity of the macrophages is assessed using live-cell video microscopy. CFSE fluorescent dye was used to stain the 4T1 mouse mammary carcinoma cells. The stain labels 4T1 cells to distinguish them from the cocultured M1 macrophages within a single imaging dish. RAW 264.7 macrophages are polarized with LPS and IFN-&#947; into an M1 phenotype. To ensure a complete polarization, immunostaining with anti-iNOS antibody conjugated to FITC was performed. Subsequently, a multipoint series of time-lapse images was acquired to observe multiple events, including phagocytosis, within the coculture.
A better understanding of the interactions between tumor cells and macrophages may lead to potential cancer immunotherapy. Live-cell imaging offers a detailed view of cellular dynamics in a real-time setting and has been used to study cell migration, phenotypic screening, apoptosis, and cytotoxicity8,9 in neuroscience, developmental biology, and drug discovery. Although the proposed tumor in this study is breast cancer, the method can also be applied to multiple target cells and distinct effector cells.

<table><tbody><tr><td>Anti-INOS antibody conjugated FITC</td><td>Miltenyi Biotec</td><td>REA982</td><td>150 &amp;micro;g in 1 mL</td></tr><tr><td>Carboxyfluorescein succinimidyl ester (CFSE)</td><td>Invitrogen</td><td>C1157</td><td>25 mg</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td>10 mg</td></tr><tr><td>DMEM/high glucose</td><td>HyClone</td><td>SH30003.04</td><td>670.0 g</td></tr><tr><td>Fetal bovine serum</td><td>Tico Europe</td><td>FBSEU500</td><td>500 mL</td></tr><tr><td>Lipopolysaccharides</td><td>Sigma</td><td>L4516-1MG</td><td>1 mg</td></tr><tr><td>Mouse recombinant interferon-gamma</td><td>Stemcell Technologies</td><td>78021</td><td>100 &amp;micro;g</td></tr><tr><td>Penicillin-streptomycin solution</td><td>Cellgro</td><td>30-003-CI</td><td>100 mL</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P5368-10PAK</td><td>10 pack</td></tr><tr><td>Trypsin EDTA</td><td>Cellgro</td><td>25-052-CI</td><td>1X, 100 mL</td></tr><tr><td>1000 &amp;micro;L pipette tips</td><td>WhiteBox</td><td>WB-301-01-052</td><td>5000 tips/case</td></tr><tr><td>2 mL serological pipette</td><td>JET BIOFIL</td><td>GSP012002</td><td>Non-pryogenic</td></tr><tr><td>200 &amp;micro;L pipette tips</td><td>WhiteBox</td><td>WB-301-02-302</td><td>20,000 tps/case</td></tr><tr><td>25 cm2 cell culture flask</td><td>Corning</td><td>CLS430639</td><td>Tissue culture treated</td></tr><tr><td>Bench top centrifuge</td><td>Dynamica</td><td>FA15C</td><td>Model: Velocity 14R</td></tr><tr><td>Biological safety fume hood</td><td>Nuaire</td><td>NU-565-400</td><td>Model: Home/ LabGard&amp;reg; ES TE NU-565 Class II, Type B2 Biosafety Fume Hood</td></tr><tr><td>CO2/air mixer</td><td>Chamlide (Live Cell Instrument)</td><td>FC-R-20</td><td>FC-5 (CO2/Air Mixer) with the flow meter</td></tr><tr><td>Cell scrapper</td><td>NEST</td><td>710001</td><td>220 mm</td></tr><tr><td>CO2 cell incubator</td><td>Panasonic</td><td>N/A</td><td>Model: MCO-19M(UV)</td></tr><tr><td>Confocal microscope</td><td>Nikon Instruments Inc.</td><td>N/A</td><td>Nikon Ti-Eclipse</td></tr><tr><td>Glass bottom dish</td><td>Ibidi</td><td>81218-200</td><td>35 mm</td></tr><tr><td>NIS elements software</td><td>Nikon Instruments Inc.</td><td>Available online download</td><td></td></tr><tr><td>Pipette controller</td><td>CappAid</td><td>PA-100</td><td>CappController pipette controller, 0.1-100ml</td></tr><tr><td>Thermostat</td><td>Shinko</td><td>Discontinued</td><td>JCS-33A 48 x 48 x 96.5mm</td></tr></tbody></table>

time-lapse 2d imaging of phagocytic activity in m1 macrophage-4t1 mouse mammary carcinoma cells in co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

The time-lapse two-dimensional (2D) images of the coculture model of 4T1 mouse mammary carcinoma cell lines show the 4T1 cells being engulfed by M1 macrophages during a 13 h period. It is important to ensure a complete polarization of the M1 macrophages by performing immunostaining. The results (Figure 1) show that the concentration of 100 ng/mL lipopolysaccharides (LPS) and 20 ng/mL IFN-&#947; polarized RAW 264.7 macrophages into the M1 state. Labeling the targeted cells with a fluorescent dye and leaving the effector cells unstained allows for a live-cell coculture model (Movie 1). Throughout the 13 h and 15 min interval, the phagocytic activity of the M1 macrophages in coculture with the 4T1 cells was documented (Movie 2). The six multipoint videos (A&#8722;F in Movie 2) were recorded to assess multiple events within a single 35 mm glass-bottom imaging dish. Movie 3 shows the 4T1 cells phagocytosed by M1 macrophages. Movie 4 was chosen as an example of an in-depth video filmed from this experiment and Movie 5 shows the uptake of 4T1 cells by M1 macrophages.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60281/60281fig01v2.jpg" /> 
Figure 1: Immunofluorescence staining with anti-iNOS-FITC in green, nuclei marker DAPI in blue, and enhanced versions of merged images. These panels represent RAW 264.7 macrophages (control) and M1 macrophages (LPS and IFN-&#947; stimulated) immunostained with anti-iNOS-FITC (M1 marker) in green and counterstained with nuclei marker, DAPI in blue. (A) DAPI stain can be observed both RAW 264.7 and M1 macrophages. (B) Anti-iNOS-FITC (green) only fluoresce on M1 macrophages. (C) Merged images of DAPI stain and anti-iNOS-FITC. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60281/60281fig02.jpg" /> 
Figure 2: Setting up fluorescence and DIC image acquisition in imaging software acquisition dialog control. [1] select the [Lambda] tab checkbox, [2] select [Optical Configuration] and select [10X DIC] and [GFP-R] for the green fluorescence filter checkbox, [3] select [10X DIC] | [Set this channel as the focus reference]. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60281/60281fig03.jpg" /> 
Figure 3: Setting up multipoint image acquisition in the imaging software acquisition dialog control. [4] Select the [XY] tab and next [5] select the checkbox under the [Point Name] column to set each point for the image capture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60281/60281fig04.jpg" /> 
Figure 4: Setting up intervals and duration for time-lapse measurement image capture. The imaging software acquisition dialog control to set up time-lapse image capture. To set up the time-lapse image acquisition [6] check on the [Time] tab, [7] determine the [Interval] (by sec, min, or hour) and [8] the [Duration] (by sec, min, or hour). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60281/60281fig05.jpg" /> 
Figure 5: Setting up multipoint movie panels for time-lapse measurement image capture. The preview of six multipoint movies panels combined after completion of 13 h of coculture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/60281/60281vid01.jpg" /> 
Movie 1: A representative movie of unstained M1 macrophages and 4T1 CFSE-stained mouse mammary carcinoma cells in coculture captured using multichannel acquisition of fluorescent and DIC microscopy. Results showcasing CFSE in the green-labeled cell wall of 4T1 mouse mammary carcinoma cells cocultured with unstained M1 macrophages. The time-lapse images were acquired for a 13 h coculture period with 15 min time intervals using multichannel acquisition (step 7.3, see Figure 2). (A) Differential interference contrast, (red circle) shows small, round M1 macrophages adhering to the 4T1 cells, which have an epithelial morphology. (B) A fluorescence microscopy image of 4T1 cells stained with CFSE before phagocytosis by macrophages. (C) Merged DIC and fluorescence microscopy images of CFSE-stained 4T1 cells being engulfed by unstained M1 macrophages. The blue arrows show unstained M1 macrophages fluorescing green as they engulf CFSE-labeled 4T1 cells. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281_R3_Movie_1.mov" target="_blank">Please click here to download this video.</a>
<img alt="Movie 2" src="/files/ftp_upload/60281/60281vid02.jpg" /> 
Movie 2: Live-cell video microscopy movies from multiple stage points in a single imaging dish showing phagocytosis of 4T1 mouse mammary carcinoma cells by induced M1 macrophages. Results represent six different points (Movie 2A&#8722;F) set up and coordinated using imaging software (step 7.4, see Figure 3&#8722;Figure 5). M1 macrophages have an irregular, pancake-like morphology with a porous cytoplasm, whereas 4T1 mouse mammary carcinomas have an epithelial morphology. M1 macrophages and 4T1 cells were cocultured for 13 h inside the stage incubator at 37 &#176;C with 5% CO2 before being observed for live-cell video microscopy. Images were captured at 15 min time intervals for 13 h. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281_R3_Movie_2.mov" target="_blank">Please click here to download this video.</a>
<img alt="Movie 3" src="/files/ftp_upload/60281/60281vid03.jpg" /> 
Movie 3: Live-cell video microscopy movie of a single coordinate point (see Movie 2) chosen to show the phagocytosis of 4T1 mouse mammary carcinomas by induced M1 macrophages in detail. The red arrow shows phagocytosis. The yellow circle shows that the number of 4T1 cells that quenched the engulfment of 4T1 cells. Scale bar = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281_R3_Movie_3.mp4" target="_blank">Please click here to download this video.</a>
<img alt="Movie 4" src="/files/ftp_upload/60281/60281vid04.jpg" /> 
Movie 4: A detailed video to further visualize the phagocytosis process of 4T1 cells by M1 macrophages. This panel shows live M1 macrophage cells with a porous cytoplasm phenotype. Scale bar = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281_R3_Movie_4.mov" target="_blank">Please click here to download this video.</a>
<img alt="Movie 5" src="/files/ftp_upload/60281/60281vid05.jpg" /> 
Movie 5: Macrophages moving towards 4T1 cells to establish cell-to-cell contact, followed by the uptake of 4T1 cells by M1 macrophages (see Movie 2A). The phase-contrast microscopy time-lapse video was imaged in 15 min time intervals for 13 h of coculture inside a stage top incubator at 37 &#176;C with 5% CO2. Scale bar = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60281/60281_R3_Movie_5.mov" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures at JoVE.com

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

time-lapse-2d-imaging-phagocytic-activity-m1-macrophage-4t1-mouse

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.1K Views.  Universiti Putra Malaysia. Het algemene doel van deze studie is het verkrijgen van een Time-Lapse 2D Imaging van phagocytische activiteit in M1 Macrofagen 4T1 Mouse Mammary Carcinomas Cells in Co-culturen. De levenscellen co-culturen model werd vastgesteld en waargenomen met behulp van een combinatie van fluorescerende en differentiële interferentie contrast microscopie. De beoordeling van fagocytose activiteit door macrofagen waren beeld;met behulp van imaging software te verdubbelen multi point time lapse video, voo...

Video: Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Medicine

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Immunology and Infection

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

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