We thank Bruno Amati for the kind gift of the Rosa26-MycER transgenic mouse model. This work was funded by grants from WWCR, AIRC, ERC, and the Italian Ministry of Health to P.G.P. T.V. and X.A. were supported by FIRC and A.S. by a FUV grant.

In vivo procedures were performed in accordance with EU directive 2010/63 and after approval from our institutional ethics committee (Organism for Animal Wellbeing--OPBA) and the Italian Ministry of Health (IACUC Numbers 762/2015 and 537/2017).
1. Murine Mammary Gland Collection and Digestion
<ol>
	<li>For a typical experiment, sacrifice 8-10 weeks-old virgin female mice by CO2 inhalation. Depending on the aims of the experiment, use 5-30 mice. Place the mice on dissection boards under a hood. Use needles to stretch the forelimbs and wash down the fur of the animal with ethanol.</li>
	<li>Lift the skin with forceps and perform a vertical incision starting at the level of the pelvic area and moving all the way to the cervix, leaving the peritoneum intact. To avoid rupturing the skin or the peritoneum, use round-edged scissors.</li>
	<li>Carefully detach the skin from the body with gentle movements of the scissors across the lateral axis of the body.</li>
	<li>Once the skin is fully detached from the thoracic and abdominal area, perform four incisions across the four limbs of the animal and pin down the skin with needles. Use the scissors and forceps to fully detach the body of the animal from the extended skin.</li>
	<li>Use the forceps to gently lift the mammary fat pads that are now fully exposed and carefully detach them from the skin with the aid of the scissors. Collect the lower thoracic and abdominal mammary glands of each mouse and immerse them in Dulbecco&#39;s phosphate buffered saline (DPBS), in a 50 mL conical tube. Collect up to 20 glands per tube. Keep the tissues on ice. 
	NOTE: If needed, the glands can remain on ice overnight. This is a safe stopping point. The following steps should be performed under sterile conditions.</li>
	<li>Prepare and filter the digestion medium: Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 200 U/mL collagenase and 100 &#181;g/mL hyaluronidase (see Table of Materials).</li>
	<li>Transfer up to 20 glands to a 100 mm Petri dish and mince the tissue using a scalpel or curved scissors. Avoid transferring large volumes of DPBS to the dish.</li>
	<li>Add 10 mL of digestion medium to each Petri dish and transfer the minced tissue to a 50 mL conical tube, using a 25 mL serological pipette.</li>
	<li>Seal the tubes with parafilm and place them on a rotator. Set the rotator at a low speed (0.03 x g) and incubate for 2.5 h at 37 &#176;C in a humid atmosphere containing 5% CO2.</li>
	<li>Visually inspect the suspension before proceeding to the next steps. If large pieces of tissue remain undigested, prolong the incubation at 37 &#176;C for another 30 min. 
	NOTE: As an alternative, the digestion can be performed overnight at 37 &#176;C, 5% CO2, using a gentle collagenase/hyaluronidase enzyme mix13.</li>
</ol>
2. Isolation of Primary Murine MECs
<ol>
	<li>Stop the rotator and remove the tubes from the incubator. Adjust a P1000 tip at the opening of a 5 mL serological pipette. If the suspension passes through the P1000 tip, proceed to the next steps. Otherwise, prolong the incubation at 37 &#176;C for another 30 min.</li>
	<li>Centrifuge at 100 x g, for 5 min at 4 &#176;C. Carefully decant the supernatant and resuspend the pellet of each tube in 3 mL of DPBS. 
	NOTE: The low centrifugation speed allows the removal of lymphocytes and adipose tissue cells. Gentle manipulation is required to avoid dislodging the pellet at this step.</li>
	<li>Filter the cell suspension in each tube separately, using 100, 70 and 40 &#181;m cell strainers. At each filtering step, wash the strainers with 2 mL of DPBS before collecting the pass-through. 
	NOTE: From this point on, the cell suspensions can be pooled and processed together.</li>
	<li>Centrifuge at 300 x g, for 5 min at 4 &#176;C. Carefully decant the supernatant and resuspend the cells in the remaining volume of DPBS.</li>
	<li>Proceed to the red blood cell (RBC) lysis by adding an equal volume of ammonium-chloride-potassium (ACK) lysis buffer (see Table of Materials). Mix by pipetting and incubate on ice for up to 5 min.</li>
	<li>Add 10 mL of DPBS and centrifuge at 300 x g, for 5 min at 4 &#176;C. Carefully decant the supernatant and visually inspect the pellet. If the pellet is white, proceed to the next step. Otherwise repeat the RBC lysis step (step 2.5).</li>
	<li>Resuspend the cell pellet in 1-5 mL of mammosphere media: mammary epithelial cell growth basal medium (MEBM), supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 5 &#181;g/mL insulin, 0.5 &#181;g/mL hydrocortisone, 2% B27, 20 ng/mL epidermal growth factor (EGF), 20 ng/mL basic fibroblast growth factor (bFGF) and 0.4 IU/mL heparin (see Table of Materials).</li>
</ol>
3. Serial Mammosphere Re-plating
<ol>
	<li>For the establishment of mammosphere cultures, plate the cells onto non-tissue culture treated (ultra-low adhesion) 6-well plates at a density of 200,000 viable cells/mL in mammosphere medium. Incubate the cells for 7-10 days at 37 &#176;C, 5% CO2.</li>
	<li>Prepare and filter 1% poly-HEMA solution in 95% ethanol (see Table of Materials). Coat ultra-low adhesion 6-well plates for the following passages, by adding 400 &#181;L of 1% poly-HEMA solution per well and allowing the ethanol to dry completely. For better results, repeat the coating twice. 
	NOTE: The use of non-coated plates during the first passage allows the selective removal of fibroblasts from the culture.</li>
	<li>At the end of the 7-10 days culture, collect the primary mammospheres from all the wells and centrifuge at 300 x g, for 5 min at 4 &#176;C.</li>
	<li>Carefully decant the supernatant and proceed to the mechanical dissociation of the mammospheres using a P200 pipette with filtered tips. Pipette for approximately 100 times and visually inspect the suspension for the presence of large spheroids. 
	NOTE: A short incubation (2-5 min) of the sphere pellet in a low volume (0.2-0.5 mL) of trypsin or Accutase at 37 &#176;C, 5% CO2, can be used to facilitate mechanical dissociation. Prepare fresh mammosphere medium (step 2.7) for the plating of each passage.</li>
	<li>Resuspend in 1-5 mL of fresh mammosphere medium and count the viable cells. If applicable, split the cells in treatment groups or culture conditions.</li>
	<li>Plate 20,000 viable cells/mL on poly-HEMA-coated ultra-low adhesion 6-well plates and incubate at 37 &#176;C, 5% CO2. 
	NOTE: Mammospheres reach their maximum size within 5-7 days after cell plating. When possible, distribute the cells of each sample or condition to multiple wells to obtain technical replicates. Plating density should be kept low to avoid cell aggregation. A maximum of 20,000 cells/mL is recommended for 6-well plates and 5,000 cells/mL for 24-well plates.</li>
	<li>After 5-7 days, count the number of spheres per well. This can be done either manually, using a microscope equipped with a 10x magnification lens, or using digital image analysis (see step 4).</li>
	<li>Collect the mammospheres of each well separately and centrifuge at 300 x g, for 5 min at 4 &#176;C.</li>
	<li>Carefully decant the supernatant and proceed to the mechanical dissociation of the mammospheres using a P200 pipette with filtered tips. Pipette for approximately 100 times and visually inspect the suspension for the presence of large spheroids.</li>
	<li>Resuspend in 1 mL of fresh mammosphere medium and count the viable cells. Pool the cells of each sample or condition and repeat steps 3.6-3.10. Record the number of cells plated and the number of spheres and cells counted per well for each passage. Proceed to step 5 for the cumulative growth calculation.</li>
</ol>
4. Sphere Enumeration Using Digital Image Analysis (DIA)
<ol>
	<li>At the end of each passage, scan the entire surface of all the wells and acquire images of the spheres using a digital camera mounted on a stereoscope. Save the images as .tif files.</li>
	<li>Import the stereoscope images as an Image Sequence using ImageJ14. Set the scale and the type of image to 8-bit.</li>
	<li>Duplicate the stack of images and select Subtract Background from the tab Process on the menu bar. Check the options Light background and Sliding paraboloid and click on the button OK. Process all the images of the stack.</li>
	<li>Select Adjust and then Threshold from the tab Image of the menu bar. By clicking on Apply, a dialogue window titled Convert Stack to Binary will appear. Select Default as method and Light as background. Check the box Calculate threshold for each image and click on the button OK.</li>
	<li>Select sequentially the commands Watershed, Open and Erode from the Binary list under the tab Process of the menu bar. Process all images of the stack by clicking on the button Yes of the dialogue box that appears. 
	NOTE: These processes allow for visual segmentation of objects that touch, object smoothing and removal of pixels from the edge of the objects.</li>
	<li>Select the function Analyze Particles from the menu Analyze. Set the minimum size threshold at 10,000 &#181;m2 and circularity between 0.50 and 1.00. Select the option Ellipses from the Show drop-down menu. Check Summarize, Exclude on edges and In situ Show and click on the button OK. Process all images of the stack.</li>
	<li>Visually inspect the correspondence of the ellipses with spheres and, if needed, correct the particle count accordingly. Sum the counts from all the frames of each well to obtain the total count of mammospheres per well.</li>
</ol>
5. Cumulative Growth Curve Calculation
NOTE: The number of mammospheres counted in each well at the end of each passage (PN) reflects the number of mammosphere-initiating cells seeded at the beginning of PN.
<ol>
	<li>For each passage (PN), register the number of plated cells and the number of cells and spheres counted per well. Calculate the sphere size at the end of each passage: 
	sphere size PN (cells / sphere) = cells counted PN / spheres counted PN 
	NOTE: The sphere size at PN is a measure of the proliferative potential of each mammosphere-initiating cell seeded at PN.</li>
	<li>Infer the number of plated spheres by dividing the number of cells plated for PN by the sphere size calculated at the end of the previous passage (PN-1): 
	spheres plated PN = cells plated PN / sphere size PN-1 
	NOTE: By convention, the sphere size is assumed stable during the first passage of the culture (i.e., sphere size P0 = sphere size P1). If multiple wells are used as technical replicates, use the average sphere size at PN-1 as denominator.</li>
	<li>Calculate the cumulative cell and sphere number for each well per passage: 
	cumulative number PN = (count PN / plated PN) X cumulative number PN-1. 
	NOTE: By convention, cumulative number P0 = plated P1. If multiple wells are used as technical replicates, calculate the average cumulative number per sample or condition for each passage.</li>
	<li>Plot the data points on a semi-logarithmic scale. Display the passage number (P0 to PN) on the x axis (linear scale) and the cumulative cell or sphere number on the y axis (logarithmic scale).</li>
	<li>Fit an exponential trend-line to the data points and calculate the coefficient of determination (R2) to measure the goodness of the fit. 
	NOTE: The trend-line fitted to the data points should approximate an exponential curve, as expected for a cell population that grows or dies with a constant rate. R2 takes values between 0 and 1, with values closest to 1 indicating a better fit.</li>
	<li>Depict the equation of the trend-line as a natural exponential function to infer the growth rate (GR) of the culture: 
	y = y0 e(GR)x, where y0 is the value of y when x = 0.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+entire+functional+mammary+gland+may+comprise+the+progeny+from+a+single+cell.">An entire functional mammary gland may comprise the progeny from a single cell.</a> Development. 125 (10), 1921-1930 (1998).</li><li>Shackleton, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+functional+mammary+gland+from+a+single+stem+cell.">Generation of a functional mammary gland from a single stem cell.</a> Nature. 439 (7072), 84-88 (2006).</li><li>Stingl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+unique+properties+of+mammary+epithelial+stem+cells.">Purification and unique properties of mammary epithelial stem cells.</a> Nature. 439 (7079), 993-997 (2006).</li><li>van Amerongen, R., Bowman, A. 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The authors have nothing to disclose.

Here, we describe a protocol for the quantitative description of MaSC growth properties in vitro. As an example, we present the effect of low-level constitutive Myc expression on normal murine MaSCs. This approach, however, can be equally applied to various contexts. Human or murine primary cells, as well as established cell lines, can be cultured in anchorage independent conditions to establish mammosphere cultures that can be serially passaged. Gene overexpression and RNA interference can be easily introduced in the protocol with the addition of a viral transduction step at the end of the first passage (after step 3.5). Alternatively, cells can be infected in adhesion and then plated as mammospheres.
A critical aspect of the assay presented here is the seeding cell density, which should be low enough to avoid the generation of aggregates interfering with the interpretation of the results16,17. The morphology of the mammospheres can be informative to resolve this ambiguity. Only compact, round spheres should be enumerated at the end of each passage. Both the circularity of the spheroids and the size should be taken into consideration. Using the automated process of the DIA, this step is ensured with the appropriate thresholds in an objective and absolute manner. Often, progenitors will form acinar structures or smaller clusters of cells which should be excluded from the mammosphere counts. As a rule of thumb, we use a threshold of 100 &#181;m diameter. Finally, care should be taken to avoid the transfer of intact or non-fully dissociated mammopsheres from one passage to the next. On the other hand, excess pipetting will lead to increased cell death. Thus, if such difficulties are encountered, we recommend using mild trypsinization or Accutase treatment and passing the dissociated spheres through a 40 &#181;m strainer to ensure the generation of single-cell suspensions.
Sphere forming efficiency (SFE) has been used alternatively, as a surrogate for SC or CSC quantitation ex vivo in mammosphere cultures. SFE is indeed a measure of stem-like cells in a given cell population. However, it represents a less conscientious approach since it provides information only at distinct time points. The calculation of cumulative sphere numbers and the generation of cumulative growth curves, instead, enables the inference of the growth rate of the culture from the initial cell seeding step until the culture exhausts or, in the case of immortalized cultures, for the desired number of passages. The assessment of growth properties allows the evaluation of the deviation from the exponential growth through the coefficient R2 and, at a second step, the assessment of the GR value itself.
Importantly, cumulative mammosphere growth curves can be used to evaluate the effect of small molecule inhibitors or other chemotherapeutic drugs selectively at the CSC level6,11. Contrary to normal primary mammospheres, which functionally exhaust in 5-7 passages, tumor mammospheres tend to expand indefinitely. This feature is linked to the unlimited CSC self-renewal ability. The effects on proliferation and CSC self-renewal can be uncoupled through the generation of tumor cell and mammosphere growth curves, respectively. A CSC-specific effect is expected to result in a decrease in the cumulative mammosphere growth rate, with or without effect on the cumulative cell growth rate6,11.
Finally, another area of interest is the one of adult tissue SC reprogramming. Fully grown mammospheres consist of a phenotypically heterogeneous cell population, in which only a minor fraction retains stem-like features, including mammosphere-initiating ability and mammary gland regeneration upon transplantation in vivo6,9,11,18,19. Mammary progenitors can be thus isolated either using in vitro label-retaining assays6,9,11 or, ex vivo, using established surface markers2,3. Notably, mammary progenitors do not survive anoikis and are unable to form mammospheres. Enforced Myc expression has been shown to confer mammosphere initiation potential to mammary progenitors isolated as PKHneg11, resulting in the generation of a culture that can be passaged indefinitely. Similarly, interference of negative regulators of physiological reprogramming can be tested using the same assay. In this context, a common issue that may arise is the limited number of cell input. If the cell input is lower than 10,000 cells, we recommend seeding in 24-well plates (maximum 5,000 viable cells/mL). Nevertheless, anchorage independent culture conditions can be proven to be too harsh for scoring reprogramming, especially in cases where the reprogramming effect is not immediate. In such cases, the use of a supportive matrix and 3-dimensional organoid cultures could be more appropriate20.
Overall, the mammosphere assay is a cost-effective option that can be easily employed for scoring stem-like properties in normal and tumoral MEC populations. The quantitative approach taken in this protocol facilitates the comparisons between cultures carried out in different conditions or exposed to diverse stimuli. When followed rigorously, it provides a relatively simple ex vivo model system that permits uncoupling of the multiple players that define stem properties in vivo, offering the possibility of more detailed mechanistic studies.

Isolation and in vitro culture of mammary epithelial stem and progenitor cells have become essential for understanding their properties in mammary cell biology. Elegant lineage tracing and serial transplantation assays have enabled the study of stem cells (SCs) and other tissue subsets in the context of their in vivo niche. However, this approach is time consuming and requires the generation of reporter mouse models1,2,3,4,5. Therefore, in vitro culture and propagation of mammary stem cells (MaSCs) while sparing key stemness features, namely self-renewal and differentiation ability, is one of the biggest challenges in the field. In the last years, the mammosphere assay has been widely used to model both normal mammary tissue and breast cancer growth, to quantify normal or cancer SCs (CSCs) and assess their self-renewal ability as a surrogate reporter of their activity in their respective in vivo context6,7,8,9,10,11.
The mammosphere assay is an efficient and cost-effective approach, in which freshly isolated mammary epithelial cells (MECs) are cultured in non-adherent conditions, with the premise that only MaSCs will survive and form spheres in suspension while all the other cell types will die by anoikis. Moreover, the ability to form several generations of mammospheres in serial non-adherent passages is related to the self-renewal ability of the MaSCs6,9,11. Here, we describe a detailed protocol of a quantitative mammosphere assay, which was initially developed by Dontu and colleagues7 as a modification of the pioneering neurosphere assay12, enabling the growth of putative SCs in non-adherent, serum-free conditions with the addition of appropriate growth factors7,12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACK lysis buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>Ammonium-chloride-potassium lysis bufer, 100 mL.</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>B27 supplement 50X (10 mL). Final concentration 2% v/v.</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Human recombinant fibroblast growth factor - basic, 50 &#956;g. Stock solution 100 &#956;g/&#956;L in Tris 5 mM pH 7.6. Final dilution 0.02% v/v.</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C2674</td>
			<td>Collagenase from Clostridium histolyticum. Type I-A, lyophilized powder, 1 g. Stock 20,000 U/mL in DMEM. Final dilution 1% v/v.</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Microgem</td>
			<td>S17859L0615</td>
			<td>Dulbecco&#39;s phosphate buffered saline</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Tebu-Bio</td>
			<td>AF-100-15</td>
			<td>Recombinant human epithelial growth factor. Stock solution 100 &#956;g/mL in sterile dH2O. Final dilution 0.02% v/v.</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Lonza</td>
			<td>17-605E</td>
			<td>L-Glutamine, 200 mM. Final dilution 1% v/v.</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>PharmaTex</td>
			<td>34692032</td>
			<td>Stock concentration 5,000 IU/mL. Final dilution 0.008% v/v.</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H4272</td>
			<td>Type IV-S, powder, 750-3,000 U/mg solid, 30 mg. Stock solution 10 mg/mL in sterile dH2O. Final dilution 1% v/v.</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0888</td>
			<td>Stock concentration 100 &#956;g/mL. Final dilution 0.5% v/v.</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>SAFCBiosciences</td>
			<td>91077C</td>
			<td>Insulin, human recombinant, dry powder, 250 mg. Stock concentration 1 mg/mL. Final dilution 0.5% v/v.</td>
		</tr>
		<tr>
			<td>Low attachment 6-well plates</td>
			<td>Corning</td>
			<td>351146</td>
			<td>Sterile 6-well not treated cell culture plates with clear flat bottom and lid.</td>
		</tr>
		<tr>
			<td>MEBM</td>
			<td>Lonza</td>
			<td>CC-3151</td>
			<td>Mammary epithelial cell growth basal medium</td>
		</tr>
		<tr>
			<td>Penicllin-Streptomycin mixture</td>
			<td>Lonza</td>
			<td>17-602F</td>
			<td>Contains 10,000 U potassium penicillin and 10,000 &#181;g streptomycin sulfate per mL in 0.85% saline. Final dilution 1% v/v.</td>
		</tr>
		<tr>
			<td>Poly-HEMA</td>
			<td>Sigma</td>
			<td>P3932</td>
			<td>Dissolve in 95% EtOH overnight at 55 &#176;C. Stock concentration 12% w/v. Final dilution 1% v/v in 95% EtOH. Filter (0.22 &#956;m) before use.</td>
		</tr>
	</tbody>
</table>

quantification of self-renewal in murine mammosphere cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Myc overexpression in normal MECs, leads to an increased frequency of mammosphere initiating cells. This is achieved through a double mechanism: Myc increases the rate of MaSC symmetric divisions and the frequency of progenitor reprogramming into new MaSCs11. To test the effect of low constitutive Myc expression, we used the Rosa26-MycER transgenic mouse model, in which Myc activity can be induced by 4-hydroxytamoxifen (4-OHT)15. We first plated the MECs on ultra-low adhesion 6-well plates to remove fibroblasts, in the absence of 4-OHT. After the first passage, we split the culture in two: two wells were kept untreated (control) and two wells were treated with 200 nM 4-OHT (MycER). We counted the sphere and cell numbers of 5 consecutive passages for three independent experiments (Table 1). The cumulative cell and sphere numbers per passage are shown on Table 2. Induction with 4-OHT leads to increased sphere and cell growth rates, as shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60256/60256fig1a.jpg" /> 
Figure 1: Representative results. Cumulative sphere (A) and cell (B) growth curves of control and MycER mammospheres. Mean and standard deviation of 3 independent experiments are shown. <a href="https://www.jove.com/files/ftp_upload/60256/60256fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="3">1st plating</td>
			<td colspan="3">2nd plating</td>
			<td colspan="3">3rd plating</td>
			<td colspan="3">4th plating</td>
			<td colspan="3">5th plating</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
			<td>Cells Plated</td>
			<td>Cell Count</td>
			<td>Sphere Count</td>
		</tr>
		<tr>
			<td rowspan="4">Exp1</td>
			<td rowspan="2">Control</td>
			<td>77,000</td>
			<td>50,000</td>
			<td>31</td>
			<td>65,000</td>
			<td>58,500</td>
			<td>41</td>
			<td>57,000</td>
			<td>30,000</td>
			<td>32</td>
			<td>30,000</td>
			<td>22,000</td>
			<td>5</td>
			<td>22,000</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>77,000</td>
			<td>81,000</td>
			<td>41</td>
			<td>65,000</td>
			<td>55,000</td>
			<td>23</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">MycER</td>
			<td>77,000</td>
			<td>31,0000</td>
			<td>193</td>
			<td>80,000</td>
			<td>375,000</td>
			<td>323</td>
			<td>80,000</td>
			<td>380,000</td>
			<td>217</td>
			<td>80,000</td>
			<td>220,000</td>
			<td>223</td>
			<td>80,000</td>
			<td>170,000</td>
			<td>155</td>
		</tr>
		<tr>
			<td>77,000</td>
			<td>110,000</td>
			<td>142</td>
			<td>80,000</td>
			<td>505,000</td>
			<td>396</td>
			<td>80,000</td>
			<td>270,000</td>
			<td>149</td>
			<td>80,000</td>
			<td>290,000</td>
			<td>194</td>
			<td>80,000</td>
			<td>250,000</td>
			<td>160</td>
		</tr>
		<tr>
			<td rowspan="4">Exp2</td>
			<td rowspan="2">Control</td>
			<td>75,000</td>
			<td>75,000</td>
			<td>71</td>
			<td>60,000</td>
			<td>17,000</td>
			<td>34</td>
			<td>28,000</td>
			<td>45,000</td>
			<td>29</td>
			<td>45,000</td>
			<td>13,000</td>
			<td>2</td>
			<td>13,000</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>75,000</td>
			<td>47,000</td>
			<td>45</td>
			<td>60,000</td>
			<td>11,000</td>
			<td>47</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">MycER</td>
			<td>75,000</td>
			<td>200,000</td>
			<td>188</td>
			<td>80,000</td>
			<td>225,000</td>
			<td>277</td>
			<td>80,000</td>
			<td>230,000</td>
			<td>155</td>
			<td>80,000</td>
			<td>210,000</td>
			<td>211</td>
			<td>80,000</td>
			<td>100,000</td>
			<td>95</td>
		</tr>
		<tr>
			<td>75,000</td>
			<td>250,000</td>
			<td>192</td>
			<td>80,000</td>
			<td>202,500</td>
			<td>283</td>
			<td>80,000</td>
			<td>305,000</td>
			<td>185</td>
			<td>80,000</td>
			<td>160,000</td>
			<td>237</td>
			<td>80,000</td>
			<td>100,000</td>
			<td>133</td>
		</tr>
		<tr>
			<td rowspan="4">Exp3</td>
			<td rowspan="2">Control</td>
			<td>82,500</td>
			<td>130,000</td>
			<td>121</td>
			<td>80,000</td>
			<td>45,000</td>
			<td>105</td>
			<td>80,000</td>
			<td>110,000</td>
			<td>86</td>
			<td>80,000</td>
			<td>58,500</td>
			<td>75</td>
			<td>58,500</td>
			<td>58,500</td>
			<td>78</td>
		</tr>
		<tr>
			<td>82,500</td>
			<td>125,000</td>
			<td>177</td>
			<td>80,000</td>
			<td>71,250</td>
			<td>42</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">MycER</td>
			<td>82,500</td>
			<td>325,000</td>
			<td>457</td>
			<td>80,000</td>
			<td>610,000</td>
			<td>327</td>
			<td>80,000</td>
			<td>367,000</td>
			<td>309</td>
			<td>80,000</td>
			<td>500,000</td>
			<td>260</td>
			<td>80,000</td>
			<td>115,000</td>
			<td>146</td>
		</tr>
		<tr>
			<td>82,500</td>
			<td>475,000</td>
			<td>463</td>
			<td>80,000</td>
			<td>455,000</td>
			<td>392</td>
			<td>80,000</td>
			<td>415,000</td>
			<td>204</td>
			<td>80,000</td>
			<td>470,000</td>
			<td>295</td>
			<td>80,000</td>
			<td>185,000</td>
			<td>161</td>
		</tr>
	</tbody>
</table>
Table 1: Numbers of spheres counted and numbers of cells plated and counted at each passage.
<img alt="Table 2" class="xfigimg" src="/files/ftp_upload/60256/60256table2.jpg" /> 
Table 2: Calculation of plated sphere numbers and cumulative sphere and cell numbers. <a href="https://www.jove.com/files/ftp_upload/60256/Table2.xlsx" target="_blank">Please click here to view a larger version of this table. (Right-click to download.)</a>

Watch this Scientific Journal Video about Quantification of Self-renewal in Murine Mammosphere Cultures at JoVE.com

Quantification of Self-renewal in Murine Mammosphere Cultures

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

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6.2K Views.  European Institute of Oncology IRCCS. Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

Video: Quantification of Self-renewal in Murine Mammosphere Cultures

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Quantification of  Colonic Stem Cell Mutations

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 &#181;m thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 &#181;m apart were prepared to assess a total of &#62;104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts &#247; by the number of wild type (blue) crypts in a treatment group.

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...