This work is supported by the Joseph Alexander Foundation, the ASBMB Undergraduate Research Award, 2013-2014, and a Science Award Grant, Marymount Manhattan College, 2012-2013.

Notes: Conduct all work with cells and cell culture reagents in a Class II biosafety cabinet (BSC)9. MMT cells are classified as Biosafety Level I, as they pose low to moderate biological risk. Apply proper cleaning and decontamination procedures to the BSC between uses (e.g., ultraviolet light, 70% ethanol wipe down).
1. Grow MMT cells
<ol>
	<li>Grow cells in 10 cm tissue culture dishes containing 10 ml of nutrient rich media that consists of Eagles Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 1% Antimycotic/Antibiotic (10 units penicillin, 100 &#181;g streptomycin, 0.25 &#181;g amphotericin B). Cultivate cells in a humidified 5% CO2 chamber, at 37 &#176;C. Plate cells at a density of 3.6 x 106 cells/cm2 (see Counting Cells in step 2).</li>
	<li>Replace half of the cell culture media with fresh media every 48 hrs unless otherwise indicated. Check cell viability and morphology by examination with an inverted phase contrast light microscope.
	<ol>
		<li>Note and characterize cells for size, structure, shape, organization and estimated number under at 100 - 200X magnification. At this density, the cells should appear in one plane, not clumped atop each other and in close proximity. Each cell consists of a thick, spherical core with thin, long, branch-like extensions expanding from it that come to a point (see Figure 1).</li>
	</ol>
	</li>
	<li>Subdivide cells when they reach a cell density of 7.2 x 106 cells/cm2. Cells exhibit a doubling rate of 1.8 x 106 cells/cm2 per 24 hrs. Designate cells Passage X (Px) to denote the number of times they have been split.
	<ol>
		<li>At generation 3 (i.e., after three passages, P3), harvest, count, and plate the cells onto a 24 well tissue culture plate at a density of 3.6 x 106 cells/cm2.</li>
	</ol>
	</li>
	<li>View cells every day and record digital photomicrographs. Note and describe cell morphology (i.e., size, shape, light reflective properties). Determine cell doubling time by counting cells regularly and relating elapsed time to cell number.</li>
</ol>
2. Count MMT Cells
Note: Count cells to determine if cells need to be subcultured, to set up for an experiment or to determine cell viability. There are two methods presented here.
<ol>
	<li>Use of a hemocytometer, a standard technique to count cells.
	<ol>
		<li>Obtain a bright-line hemocytometer, a 0.2% solution of trypan blue dye, a compound light microscope, sterile test tubes, pipettes and a manual cell counter.</li>
		<li>Under the BSC, use a 10 ml pipette to remove cells from a 10 cm tissue culture dish. If cells are grown on a 24 well plate use a 1 ml pipette or 1,000 &#181;l micropipette to remove the media.</li>
		<li>Draw cell media into the pipette, place the tip of the pipette against the bottom of the dish and expel the media while sliding the pipette tip across the plate. Repeat 4 - 5 times to help dislodge cells, dissociate clumps and lead to a more accurate cell count.</li>
		<li>Place the resulting cell suspension in a 15 ml sterile tube (or 1 ml micro centrifuge or other small volume tube). Otherwise, leave the cells in the original tissue culture plate/well.</li>
		<li>Under the BSC, combine 10 &#181;l of the cell suspension with 10 &#181;l of the trypan blue solution (1:1 ratio) in a non-sterile micro centrifuge or other small volume tube. 
		Note: Trypan blue is a vital stain that is not absorbed by healthy viable cells. When cells are damaged or dead, trypan blue can enter the cell allowing dead cells to be counted (aka dye exclusion method). Therefore under a microscope, dead cells appear dark purple while viable cells are a bright and light in color.</li>
		<li>Incubate at RT for 5 min.</li>
		<li>Place the cover slip on the hemocytometer. Apply 10 &#181;l of the trypan blue-cell suspension mixture into the groove. View the hemocytometer at 100 - 200X magnification. A four-corner grid is apparent (see Figure 2).</li>
		<li>Count the number of viable cells (unstained) versus total cells in each gridded area. Average the four grids and multiply by 1 x 104 to obtain cells/ml.
		<ol>
			<li>Extrapolate the total number of cells per dish (or cells/cm2). Use this number to either determine the correct volume of the cell suspension to use to seed a new tissue culture plate at the desired density of 3.6 x 106 cells/cm2&#160;or determine total number of viable cells on the plate or in the well. 
			Note: For additional guidance on the use of a hemocytometer, see10.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Use software and a digital camera to count viable cells.
	<ol>
		<li>Obtain a digital camera, its accompanying software, a 0.4% solution of trypan blue dye, a compound light microscope, sterile test tubes, pipettes and a computer. 
		Note: A Moticam 2,000 digital camera with accompanying Motic Software is employed here. Any comparable camera and software should be sufficient. Ensure that the digital camera software has been calibrated in advance (according to manufacturer&#8217;s protocol).</li>
		<li>Under the BSC, use a 1 ml pipet or 1,000 &#181;l micropipette to remove the media from the MMT cells growing in a 24 well tissue culture dish. Wash the adherent cells by gently applying a small amount (approximately 500 &#181;l) of un-supplemented (i.e., EMEM without any supplements) fresh media to the plate then removing it. Apply 10 &#181;l of 0.2% trypan blue (made by diluting 1:1 with un-supplemented EMEM) directly to the well.</li>
		<li>Incubate the plate at RT for 5 min.</li>
		<li>View the plate under the microscope at 100 - 200X magnification with a digital camera attached.&#160;Plate may be washed with unsupplemented media if staining with 2% trypan blue is too dark.</li>
		<li>Open the software on the computer and verify that the software is calibrated to the objective used via the settings tab. An image of the FOV should appear.</li>
		<li>Select the Grid from the Measure tab. A grid of squares approximately 0.005 cm in width will appear. Select Grid Info to confirm the area of each square.</li>
		<li>Choose a specific area of the grid to count cells (i.e., 5 squares x 9 squares). Select Rectangle from the Measure tab. Click the corner of a square and drag your cursor to encompass the squares of choice. 
		Note: A shade of green will cover the squares of choice and a white box will appear in the corner that provides the width, height, area, and perimeter of the section of the grid chosen (see Figure 3).</li>
		<li>Count the number of viable cells within the determined area. Do the same for two other locations in the same well or plate by moving the plate under the microscope.</li>
		<li>Be sure that the measured area is the same and the magnification has not changed. Calculate the average number of viable cells within the area. Using the area of the well (for a 24 well plate, one well has an area of 2 cm2, for a 100 mM dish the area is 78.6 cm2), extrapolate the number of viable cells from the area delineated in the grid to the total number of cells within the well (cells/cm2).</li>
	</ol>
	</li>
</ol>
3. Treat MMT Cells with anti-proliferative Agents
<ol>
	<li>Prepare solutions of the selected anti-proliferative therapeutic agents (tamoxifen,&#160;curcumin and metformin) and optional drug, aspirin under the BSC.
	<ol>
		<li>Dissolve curcumin and tamoxifen in 100% ethanol to generate a stock concentration of 27 mM. Dissolve metformin and aspirin in unsupplemented EMEM to generate a stock concentration of 500 mM and 15 mM, respectively.</li>
	</ol>
	</li>
	<li>Establish a Dose Response.
	<ol>
		<li>Treat MMT cells with the three anti-proliferative therapeutic agents (tamoxifen, curcumin and metformin) and optional drug (aspirin) at varying concentrations for 96 hrs to generate a dose response curve. Initially administer all drugs at a range of concentrations based on published reports1,11-16 and then at concentrations larger or smaller than those published. 
		Note: A dose response determines the minimum concentration of a drug necessary to produce the desired results. Here the desired result is a reduction in cell proliferation as compared to the control.
		<ol>
			<li>For tamoxifen and curcumin, use concentrations (and corresponding volumes) of 0.054 mM (1 &#181;l), 0.108 mM (2 &#181;l), 0.162 mM (3 &#181;l) and 0.216 mM (4 &#181;l).</li>
			<li>For metformin, use concentrations (and corresponding volumes) of 2 mM (2 &#181;l), 4 mM (4 &#181;l), 6 mM (6 &#181;l), 8 mM (8 &#181;l) and 10 mM (10 &#181;l).</li>
			<li>For aspirin, use concentrations (and corresponding volumes) of 0.030 mM (1 &#181;l), 0.060 mM (2 &#181;l), 0.099 mM (3.3 &#181;l), 0.150 mM (5 &#181;l), and 0.216 mM (6.7 &#181;l).</li>
		</ol>
		</li>
		<li>Split MMT cells from the 10 cm dish onto a 24 well plate at a concentration of 3.6 x 106 cells/cm2. Determine initial cell concentration by both cell-counting methods (Step 2). Call this new 24 well plate of cells &#8220;Day Split&#8221;. </li>
		<li>24 hrs after cell plating, treat the MMT cells with each of the anti-proliferative therapeutic agents, tamoxifen, curcumin, metformin and aspirin, at the concentrations described in Step 3.2.1. Refer to this as &#8220;Day 0&#8221;.
		<ol>
			<li>As each well can hold a maximum of 500 &#181;l of media, use micropipettes with sterile micropipette tips and a new tip each time a new well is treated to avoid cross contamination. Use two wells as controls: cells grown in the absence of any drug treatment (negative control) and cells grown in the presence 100% ethanol (solvent control).</li>
		</ol>
		</li>
		<li>Grow cells and re-administer drugs at the described concentrations when the cells are fed every other day (see Step 1.2) for 96 hrs (until Day 4). On Days 1 - 4 of treatment, observe the cells under the microscope and count using the method in Step 2.2 (see Figure 4).</li>
		<li>Repeat the experiment at least three times.</li>
		<li>Determine optimal concentration of each drug by graphing the relationship between cell viability and drug dosage over the length of the experiment (see Figure 5).</li>
	</ol>
	</li>
	<li>Establish a Time Course.
	<ol>
		<li>Treat MMT cells with the three anti-proliferative therapeutic agents (tamoxifen, curcumin and metformin) at a fixed concentration for varying time periods. Use the optimal concentration identified through the Dose Response experiments (see Step 3.2).
		<ol>
			<li>Use the following concentrations: 0.216 mM tamoxifen, 0.216 mM curcumin and 10 mM metformin. 
			Note: A time course determines the amount of time necessary for a drug to produce its optimal desired result. Here, the desired result is a reduction in cell proliferation as compared to the control.</li>
		</ol>
		</li>
		<li>Split MMT cells from the 10 cm dish onto a 24 well plate at a concentration of 3.6 x 106 cells/cm2. Determine initial cell concentration by both cell-counting methods (Step 2). Call this new 24 well plate of cells &#8220;Day Split&#8221;. </li>
		<li>24&#160;hrs after cell plating, treat the MMT cells with each of the anti-proliferative therapeutic agents, tamoxifen, curcumin, metformin and aspirin, at the optimal concentrations identified in Step 3.2. Refer to this as &#8220;Day 0&#8221;.
		<ol>
			<li>As each well can hold a maximum of 500 &#181;l of media, use micropipettes with sterile micropipette tips and a new tip each time a new well is treated to avoid cross contamination.</li>
			<li>Use two wells as controls: cells grown in the absence of any drug treatment (negative control) and cells grown in the presence 100% ethanol (solvent control).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Grow cells and re-administer drugs at the selected concentration when the cells are fed every other day (see Step 1.2) for 96 hrs (until Day 4). On Days 1 - 4 of treatment, observe the cells under the microscope and count using the method in Step 2.2 (see Figure 4).</li>
	<li>Repeat the experiment at least three times.</li>
	<li>Determine optimal exposure time of MMT cells to each drug by graphing the relationship between cell viability and length (time) of drug treatment at the selected concentration (see Figure 6).</li>
</ol>
4. The Lab Module
Note: The following is a 5-day lab schedule for the lab module. Figure 7 is a flow chart of what the schedule would entail within the 5 days. For this activity to be completed in five days either a time course or a dose response curve is generated. There is not sufficient time to generate both curves. A dose response experiment is described below. A time course experiment can be easily interchanged
<ol>
	<li>Split the class into groups: have one group establish a dose response and the other a time course choosing a concentration in the middle of the proposed dose response concentrations.
	<ol>
		<li>Maintain sufficient cultures and drug stocks at appropriate concentrations. Unless otherwise noted, perform all work under the BSC.</li>
	</ol>
	</li>
	<li>Day 1
	<ol>
		<li>Direct students to make media and grow cells (as in step 1).</li>
		<li>As students obtain a stock of MMT cells on a 10 cm plate, direct the students to prepare cell culture media and give a tutorial in the use of the hemocytometer, digital microscopy and the digital camera microscopy software (as in step 2).</li>
		<li>Calibrate the software to the objectives used on the microscope (e.g., 4X, 10X, 20X) now using manufacturer&#8217;s protocol.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Have students confirm total cell number and cell viability (as in step 2).</li>
		<li>Have students count cells on the 100 mm dish using both the microscopy software and the hemocytometer methods, to confirm similar results are obtained.</li>
		<li>Direct students to obtain a 24 well plate and split cells from the 100 mm dish to the 24 well plates to the desired starting cell plating density (as in step 1). This is considered Day Split. Place the 24 well plate in a cell culture incubator for 24 hrs.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Grow MMT cells for 24 hrs on a 24 well plate. Have students observe cells under microscope and count using microscopy software (as in step 2).</li>
		<li>Give the student/team samples of cancer treatment drug stocks. Have them prepare and dilute the original stock solution (as in step 3). Add the various concentrations of the drugs to their cells to establish a dose response curve. This is called Day 0.</li>
		<li>Incubate cells (step 1.2) with the drugs for maximum of 48 hrs.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Have students observe treated cells after 24 hrs. This is Day 1.</li>
		<li>Count each well using microscopy software. Record photographs of each well so that counting can be conducted outside the lab (as in step 2).</li>
		<li>Incubate cells (step 1.2) with the drugs for another 24 hrs. 
		Note: Instructor provides additional tutorials and reviews of data analysis and graphical presentation. Students learn to create figures, plots, and statistical analysis for the following day of data collection.</li>
	</ol>
	</li>
	<li>Day 5
	<ol>
		<li>Have students treated cells after 48 hrs, Day 2.</li>
		<li>Count each well using microscopy software. Record photographs of each well so that counting can be conducted outside the lab (as in step 2).</li>
		<li>Harvest and collect cells for counting by the traditional hemocytometer method as a means of verifying student ability to effectively use the microscopy software method (as in step 2). 
		Note: Collect data and draw conclusions or revise hypotheses, accordingly.</li>
	</ol>
	</li>
</ol>

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The authors received no financial or comparable support from Motic.

A lab module is presented that aims to teach a variety of topics in cell biology through the advanced techniques of animal cell culture. The module achieves this by analyzing the effects of a number of anti-proliferative chemicals on the replication of cells that model human breast cancer. The primary assay relies on the fundamental technique of cell counting and introduces a novel way to count cells using microscopy software. The activities comprising the module can be conducted with instruments and equipment available ...

Often in undergraduate general biology courses, the topics of cell cycle regulation and cancer are touched upon but not explored in detail because the breadth of content in these courses leaves little time for depth. In addition, undergraduate biology students are not typically exposed to the advanced techniques associated with animal cell culture. To help students develop a deeper understanding of these concepts, while applying and analyzing what they have learned, a laboratory activity was developed as a modification of the Walter Reed Army Institute of Research (WRAIR) extended laboratory activity1. The lab module uses a step-wise, experimental strategy that includes growing and characterizing a cancer cell model, developing and executing cell counting methods, establishing optimal time course and dosages for treating cells with anti-proliferative agents, and identifying aberrant cell-signaling pathways. The experiment also allows for open-ended inquiry.
Most of the techniques required for this activity can be accomplished in a typical biology-teaching laboratory. The activity starts with students characterizing the morphology and growth rate of the mouse mammary tumor (MMT) cell line, a model for human breast cancer2&#160;.&#160;Breast cancer was chosen as the model cancer because of its prevalence in the population, its familiarity to college-aged students, and the widespread data available. The MMT cell line was specifically selected because it is easily obtainable, well characterized, has a short doubling time and is easy to grow. In addition, MMT cells are estrogen-dependent which is consistent with most female breast cancers. Students then identify aberrant cell-signaling pathways in the MMT cells by treating the cells with chemotherapy drugs whose mechanism of action is well established.The concentration of the drugs and length of the treatments are varied allowing students to evaluate the effect of these variables on the rate of cell division. The key assay for this activity is the determination of cell viability, which simply requires cell counting, using one of two methods. Each method depends on strong microscopy skills. Students determine cell viability by using a standard, hemocytometer method and a novel photomicroscopy method and propose. Based on their findings, they can propose and test modifications to the activity. Students then represent their data and interpret the results to refine their hypothesis and devise new experimental strategies.
This laboratory activity is suited for freshman or sophomore level students majoring in the biological sciences. It is condensed into a one-week lab module that can be completed in a first year, general biology or second year, cellular/molecular biology course. Skills needed for proper completion of the activity include basic arithmetic and algebra, familiarity with an array of core laboratory skills (e.g., pipetting, solution making, sterile technique), data analysis, basic light microscopy and time management, along with instructor knowledge of cell culture and spreadsheet software. Reagents required include an animal cell line model for cancer (e.g., mouse mammary tumor cells, MMT2), chemotherapy agents (e.g., tamoxifen, curcumin, metformin, and aspirin), trypan blue and cell culture media (e.g., Eagle&#39;s Minimum Essential Medium; EMEM) with appropriate supplements (e.g., donor horse and fetal bovine serum). Instruments needed include an inverted light microscope with digital camera attachment, computer, 100 mm and 24&#160;well tissue culture plates, CO2 incubator (or equivalent), biosafety cabinet (BSC; Class II), hemocytometer, and digital microscopy software.
There are good examples of specific lab activities that rely on animal cell culture to teach undergraduate students about concepts in cell biology3. However many require supplies or techniques that are not easily accessible (e.g., radioactive isotopes, live animal tissue, advanced imaging equipment1,4,5), describe protocols that are quite advanced (e.g., suitable for a 400 level course6), or require multi-week or semester long projects6,7. The lab activity described here is straightforward and can be conducted in a single week with common lab equipment.
In summary, this lab module effectively introduces or reinforces the concepts of cell cycle, cellular signaling pathways and cancer while teaching basic and advanced lab skills, experimental data analysis, the method of animal cell culture and the scientific process. The laboratory module is simple and economically accessible and provides both flexibility and opportunity for open-ended inquiry. The activity encourages student creativity by providing a template experimental strategy that acts as a guide but not a recipe. Most importantly, the activity satisfies all learning domains of Blooms Taxonomy8 as it requires remembering, understanding, applying, analyzing, evaluating and creating by engaging students in a process that pulls them out of the textbook and into the world of scientific research.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>ESCO Labculture Reliant</td>
			<td></td>
			<td>Class II Type A2 Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Waterjactor CO2 Incubator</td>
			<td>CEDCO</td>
			<td>Model 1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-line Hemocytometer</td>
			<td>American Optical</td>
			<td></td>
			<td>with two separate grids</td>
		</tr>
		<tr>
			<td>Motic Images Plus</td>
			<td></td>
			<td></td>
			<td>Mac OSX Verison 2.0 or higher</td>
		</tr>
		<tr>
			<td>Gilson Pipetman</td>
			<td>Rainin instrument co. inc</td>
			<td></td>
			<td>P-20D, P-200D, P-1000D</td>
		</tr>
		<tr>
			<td>CK30/CK40 Culture Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>4 objective inverted light microscope with camera</td>
		</tr>
		<tr>
			<td>200 uL Pipet tips</td>
			<td>MidSci</td>
			<td>40200C</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 uL Pipet tips</td>
			<td>MidSci</td>
			<td>AVR4</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Seriological Pipets</td>
			<td>TPP</td>
			<td>TP94010</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>CoStar- Tissue Culture Cluster</td>
			<td align="right">3524</td>
			<td>24 wells, 16 mm well diameter, Radiation sterilized</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>100 mL, cell culture tested non-haz</td>
		</tr>
		<tr>
			<td>Bright-line Hemacytometer replacement coverslip, non-haz</td>
			<td>Sigma</td>
			<td>Z375357</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Mammary Tumor(MMT) cells</td>
			<td>ATCC</td>
			<td>CCL-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle Minimum Essentail Medium (EMEM)</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F0926</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Meformin Hydrochloride</td>
			<td>Sigma</td>
			<td>PHR1084</td>
			<td>500 mg</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>white or white-yellow powder</td>
		</tr>
		<tr>
			<td>Curmumin</td>
			<td>Sigma</td>
			<td>C1386</td>
			<td>yellow-orange powder</td>
		</tr>
		<tr>
			<td>Aspirin</td>
			<td>Sigma</td>
			<td>A2093</td>
			<td>meets USP testing specifications</td>
		</tr>
	</tbody>
</table>

using mouse mammary tumor cells to teach core biology concepts: a simple lab module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

Growing MMT cells and comparing counting methods.
Mouse mammary tumor cells were successfully grown and characterized (Figure 1) and a novel cell counting method developed using Motic Software, a digital camera-associated software program for a microscope. This new cell counting method was compared to a traditional counting method employing a hemocytometer (Figure 2) and was shown to be equally accurate in determining cell number (Table 1</str...

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Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

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13.0K Views.  Marymount Manhattan College. A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Video: Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

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Chapters in this video

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

Mammary Epithelial Transplant Procedure

Flow Cytometry Purification of Mouse Meiotic Cells

Isolation of Immune Cells from Primary Tumors

Generation of Mice Derived from Induced Pluripotent Stem Cells

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

Measuring Lactase Enzymatic Activity in the Teaching Lab