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Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946, DOI 10.1007/978-1-62703-128-8_25, © Springer Science+Business Media, LLC 2013

Chapter 25

Enzymatic Dissociation, Flow Cytometric Analysis, and Culture of Normal Mouse Mammary Tissue

Michael Prater *, Mona Shehata* , Christine J. Watson , and John Stingl

Abstract

Evidence is emerging that the mouse mammary epithelium is arranged as a hierarchy that spans from stem cells to lineage-restricted progenitor cells to differentiated luminal and myoepithelial cells. The use of fl uorescence-activated cell sorting (FACS) in combination with quantitative functional clonal assays represents a powerful tool for studying the properties of mouse mammary stem and progenitor cells. This chapter outlines the experimental procedures for generating single viable cell suspensions of mouse mammary epithelial cells, immunostaining cells for fl ow cytometry, in vitro assays for the detection and enumeration of mouse mammary progenitor cells, and in vivo assays for the detection and enumeration of mouse mammary stem cells.

Key words: Mouse mammary gland , Stem cells , Flow cytometry , Cell culture

The mouse mammary gland is a compound tubulo-alveolar gland that is composed of a series of branched ducts that drain milk pro-duced by alveolar structures during lactation. There are two gen-eral lineages of epithelial cell in the gland: luminal cells which line the lumen of the ducts and alveoli, and basal-positioned myoepi-thelial cells that reside below the luminal cells and adjacent to the basement membrane ( 1, 2 ) . Evidence suggests that distinct epithe-lial cell subtypes exist within the mammary gland and that these cells are organized as a hierarchy with the mammary stem cell at the apex of this hierarchy ( 3– 11 ) . A mammary stem cell is de fi ned

1. Introduction

*Michael Prater and Mofna Shehata contributed equally.

396 M. Prater et al.

as a cell that can generate both ducts and lobules (complete with luminal and myoepithelial cells) and can self-renew. It has been demonstrated that distinct progenitor and differentiated luminal and basal cell subpopulations exist in the mouse mammary epithe-lium ( 3– 6 ) . This chapter describes the methods to (1) dissociate mouse mammary glands, (2) generate a single cell suspension of mammary cells, (3) separate luminal, basal, progenitor, stem cell-enriched and stromal cells by fl uorescence-activated cell sorting (FACS)™, (4) detect progenitor cells using a colony-forming assay, and (5) detect mammary stem cells by their ability to generate ductal-lobular outgrowths when transplanted serially into epithe-lium-divested mammary glands of female weanling mice.

1. Dulbecco’s Modi fi ed Eagle’s Medium/Nutrient Mixture F12 Ham supplemented with 2 mM L -glutamine and 15 mM HEPES (DMEM/F12/H). Store at 4°C.

2. Collagenase and hyaluronidase enzyme dissociation mixture “Collagenase/Hyaluronidase Gentle™” (10× concentrated stock, StemCell Technologies) stored as 1 mL aliquots at −20°C.

3. Gentamicin solution (50 mg/mL, Gibco) stored at 4°C. Use at 50 m g/mL fi nal concentration.

1. Hanks’ Balanced Salt Solution (HBSS) (1×), liquid with cal-cium chloride and magnesium chloride, supplemented with 10 mM HEPES and 2% fetal bovine serum (FBS). Referred to as HF.

2. Ammonium chloride solution (NH 4 Cl, StemCell Technologies) stored at −20°C.

3. Trypsin (0.25% porcine trypsin) stored at −20°C. 4. Dispase solution 1 (5 mg/mL, StemCell Technologies) stored

as 1 mL aliquots at −20°C. 5. Deoxyribonuclease one (DNase, Sigma) dissolved at 1 mg/

mL in Dulbecco’s Modi fi ed Eagle Medium. Sterile- fi lter with a 0.22 m m fi lter. Store DNase stock (1 mg/mL) in 100 m L aliquots at −20°C.

6. Normal rat serum stored at −20°C. 7. 15 and 50 mL centrifuge tubes. 8. 5 mL FACS ™ tubes Becton Dickinson. 9. 5 mL FACS tubes with 35 m m cell strainer caps (Becton

Dickinson).

2. Materials

2.1. Dissociation of Mouse Mammary Glands

2.2. Single Cell Preparation and Staining

39725 Mouse Mammary Cell Culture

10. 40 m m cell strainer (Becton Dickinson). 11. 1.7 and 2.0 mL eppendorf tubes. 12. Antibodies and reagents for fl ow cytometry (deluted in HF):

CD45-biotin (clone 30-F11, eBioscience, use at 1 m g/mL) Ter119-biotin (clone Ter119, eBioscience, use at 1 m g/mL) CD31-biotin (clone 390, eBioscience, use at 1 m g/mL) BP1-biotin (clone 6C3 eBioscience, use at 1 m g/mL) EpCAM-Alexa Fluor (AF) 647 (clone G8.8, Biolegend, use at 1 m g/mL) CD49f-Alexa Fluor 488 (clone GoH3, Biolegend, use at 1:100 dilution) Rat IgG2a,k-Alexa Fluor 488 (clone RTK2758, Biolegend, use at 1 m g/mL) Rat IgG2a,k-Alexa Fluor 647 (clone RTK2758, Biolegend, use at 1 m g/mL) Streptavidin APC-Cy7 (Biolegend, use at 0.4 m g/mL)

13. 4 ¢ ,6-diamidino-2-phenylindole (DAPI, Invitrogen). Make up as a sterile 1 mg/mL stock solution in distilled water and store at −20°C in 1 mL aliquots. Use at 1 m g/mL fi nal concentration.

1. 60 mm cell culture dishes. 2. Mouse EpiCult-B™ basal media (StemCell Technologies)

stored at 4°C and Mouse-EpiCult-B™ supplements, stored in 5 mL aliquots at −20°C.

3. FBS (StemCell Technologies catalogue number 06100) stored in 5 mL aliquots at −20°C.

4. Recombinant human epidermal growth factor (EGF, Sigma) is dissolved at 10 m g/mL in DMEM/F12/H with 1% bovine serum albumin Fraction V (BSA, Sigma). Sterile- fi lter with a 0.22 m m fi lter. Store EGF stock (10 m g/mL) in aliquots at −20°C.

5. Recombinant human fi broblast growth factor-basic (bFGF, Peprotech) is dissolved at 10 m g/mL in DMEM/F12/H with 1% BSA. Sterile- fi lter with a 0.22 m m fi lter. Store FGF stock (10 m g/mL) in aliquots at −20°C.

6. Heparin solution (2 mg/mL, StemCell Technologies) stored at 4°C.

7. Gentamicin solution (50 mg/mL, Gibco) stored at 4°C. Use at 50 m g/mL fi nal concentration.

8. NIH 3T3 mouse embryonic fi broblasts stored in liquid nitrogen.

9. Dimethyl sulfoxide (DMSO, Fisher Scienti fi c).

2.3. Mouse Mammary Colony-Forming Assay

398 M. Prater et al.

10. Hypoxic (5% O 2 ) incubator. 11. Giemsa stain (Fisher Scienti fi c).

1. Growth factor reduced Matrigel ™ (BD Biosciences). Store at −20°C. Thaw on ice.

2. Trypan blue solution (0.4%, Sigma). 3. 25 m L Hamilton syringe 702LT Luer tip (Fisher). 4. Phosphate buffered saline (PBS). 5. BD Microlance needles, 22 Gauge (BD Biosciences). 6. Carnoy’s fi xative (60% ethanol, 30% chloroform, and 10%

glacial acetic acid). 7. Carmine (Sigma). 8. Aluminum potassium sulfate dodecahydrate (Sigma). 9. Permount Mounting Medium (Fisher Scienti fi c).

1. Prepare enzyme dissociation enzyme by adding 1 mL of 10× collagenase–hyaluronidase mixture to 9 mL DMEM/F12/H supplemented with 50 m g/mL gentamicin in a 15 mL centri-fuge tube.

2. Euthanize 1–2 virgin female mice according to institutional guidelines and then pin the mouse in a stretched position on a dissecting board and soak the abdomen with a generous amount of 70% ethanol to fl atten the fur so that the mammary glands are not contaminated upon removal ( see Fig. 1a ).

3. Make an inverted Y-shaped incision along the midsection ( see Fig. 1b ).

4. Pin back the skin ( see Fig. 1c ) and excise the fourth and third mammary glands ( see Figs. 1d–f ).

5. Add glands to the diluted collagenase–hyaluronidase solution and incubate at 37°C in a cell culture incubator for 14–16 h to digest the tissue ( see Note 1 ).

1. Remove the 15 mL centrifuge tube containing the mammary glands from the 37°C incubator and pipette with a P1000 to break up the glands (approximately 30 s).

2. Spin at 450 × g for 5 min at 4°C. 3. Discard the supernatant and suspend the cells in 1 mL of cold

HF ( see Note 2 ). 4. Add 4 mL of cold NH 4 Cl (to lyse red blood cells), mix and

spin at 450 × g for 5 min at 4°C.

2.4. Transplantation of Mammary Cells into Mouse Fat Pads

3. Methods

3.1. Dissociation of Mouse Mammary Tissue and Generation of Single Cell Suspensions

3.1.1. Removal and Dissociation of Mouse Mammary Glands

3.1.2. Preparation of a Single Cell Suspension of Mammary Cells

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Fig. 1. Dissection of the number 3 ( upper blue arrow in panel c ) and 4 mammary glands ( lower blue arrow in panel c ) from mice.

400 M. Prater et al.

5. Discard supernatant and add 1 mL of pre-warmed trypsin. Pipette gently for 1–2 min.

6. Dilute with 10 mL of cold HF and spin at 450 × g for 5 min at 4°C. 7. Gently remove the supernatant. Add 1 mL of pre-warmed dis-

pase solution and 100 m L of 1 mg/mL DNAse. Pipette gently for 1 min.

8. Add 10 mL of cold HF and fi lter through a 40 m m cell strainer.

9. Spin at 450 × g for 5 min at 4°C. 10. Cells are now ready to be stained for fl ow sorting.

1. Pre-block cells by suspending them in 1 mL of HF supple-mented with 10% normal rat serum and incubating them on ice for 10 min.

2. During the preblock incubation, remove 10 m L of the cells and add these to 10 m L trypan blue and 80 m L HF and perform a trypan blue exclusion cell count using a hemocytometer.

3. After a 10 min preblock incubation on ice, add 6 mL of HF and aliquot the cell suspension to seven FACS™ tubes at 1 mL/tube ( see Note 3 ).

4. Spin at 450 × g for 5 min at 4°C. 5. Discard supernatant, being careful to remove all liquid without

disturbing the pellet. 6. Stain cells as indicated below. Do all antibody incubations with

the cells at a cell density no greater than 10 7 cells/mL. All anti-body incubations should be done on ice for 10 min followed by one wash with 3 mL of cold HF and a centrifugation at 450 × g for 5 min at 4°C. On the last wash before addition of DAPI, fi lter the diluted cells through a 5 mL FACS™ tube with a 35 m m cell strainer cap before the centrifugation step ( see Notes 4 – 6 ). Tube 1 = unstained Tube 2 = DAPI Tube 3 = CD49f-AF488/wash/DAPI Tube 4 = EpCAM-AF647/wash/DAPI Tube 5 = CD45-biotin + CD31-biotin + Ter119-biotin + BP1-biotin/wash/streptavidin APC-Cy7/wash/DAPI Tube 6 = CD45-biotin + CD31-biotin + Ter119-biotin + BP1-biotin + isotype control-AF488 + isotype control-AF647/wash/streptavidin APC-Cy7/wash/DAPI Tube 7 = CD45-biotin + CD31-biotin + Ter119-biotin + BP1-biotin + CD49f-AF488 + EpCAM-AF647/wash/streptavidin APC-Cy7/wash/DAPI

3.2. Preparing Cells for Flow Cytometry

40125 Mouse Mammary Cell Culture

7. After the last wash, suspend all cells in cold HF supplemented with 1 m g/mL DAPI such that the fi nal density of cells in sus-pension is approximately 2 × 10 6 cells/mL.

8. Cells are now ready for fl ow cytometric analysis and/or sorting.

1. Place unstained control tube onto the FACS™ machine and run sample. Adjust voltages such that the background fl uorescence is within the fi rst log decade ( see Note 7 ).

2. Run single color control tubes adjusting for background spec-tral overlap and compensate accordingly.

3. To analyze the sample, collect at least 30,000 events. Gate around all events based on forward (FSC) and side (SSC) scat-ter, but excluding the events with the highest side scatter ( see Fig. 2a and Note 8 ). Then exclude doublets by gating the events in the FSC-height by FSC-area parameters ( see Fig. 2b ). Dead and dying cells are then excluded by gating on the DAPI-negative events and by avoiding debris using the FSC parameter ( see Fig. 2c ). Also exclude the events expressing intermediate

3.3. Flow Cytometric Analysis and Sorting of Mouse Mammary Cells

Fig. 2. Flow cytometric dot plots illustrating the gating strategy to identify viable mammary luminal and basal cells. The MRU-enriched subpopulation is highlighted by the red circle.

402 M. Prater et al.

levels of DAPI as these have low viability. To exclude most non-epithelial (termed “lineage-negative”) cells, select the events that do not express CD31, CD45, Ter119, and BP-1 ( see Fig. 2d ). Draw another plot with EpCAM on the y -axis and CD49f on the x -axis ( see Fig. 2e ), which permits visualization of the luminal (EpCAM high CD49f med ), basal (EpCAM med CD49f high ), and stromal (EpCAM - CD49f - ) cell populations ( see Note 9 ). The Mammary Repopulating Unit (MRU; operational term for a mammary stem cell)-enriched subpopulation is highlighted by the red circle ( see Note 10 ).

4. Flow sorted subpopulations of cells can be collected into 5 mL tubes containing 1–2 mL of HF ( see Note 11 ). When collect-ing lower cell numbers, an eppendorf tube containing 500 m L of HF can be used as a collection tube. When sorting very low cell numbers (<200 cells) and when accurate cell counts are required, the cells can be sorted directly into 100 m L of PBS in the wells of a 96-well ultralow adherent tissue culture dishes that have a counting grid scratched into the underside of the plate. After sorting, the plates can be spun at 450 × g for 5 min to make the cells settle and the number of cells in the well can be accurately counted under a phase contrast microscope. Add cold Matrigel to a fi nal concentration of 25% immediately after counting the cells and place the plates on ice.

1. Culture NIH 3T3 cells in high glucose Dulbecco’s Modi fi ed Eagle Medium (DMEM) supplemented with 5% FBS. When the cultures become 70–80% sub-con fl uent ( see Note 12 ), remove the media and wash once with PBS. Add pre-warmed 0.05% trypsin and incubate at 37°C with occasional agitation until the cells lift off the fl ask (about 5 min). Add an equal volume of DMEM/F12/H supplemented with 2% FBS and centrifuge at 450 × g for 5 min to pellet the cells. Suspend the cells at 1 × 10 6 cells/mL in DMEM/F12 supplemented with 2% FBS in 5 mL tubes. Irradiate the cells at 50 Grays (Gy) of gamma ( g ) ionizing irradiation (or treat with mitomycin-C if there is no access to a gamma irradiator). Centrifuge the cells at 450 × g for 5 min and discard the supernatant. Suspend the irradiated cells at a concentration of 2 × 10 6 viable cells/mL in freezing media (50% DMEM/F12 + 44% FBS + 6% DMSO). Cool at −1°C/min to −80°C and store in working aliquots of 2 × 10 6 cells per cryovial in liquid nitrogen for long-term storage.

2. Make up the mouse mammary progenitor cell culture medium: Mouse EpiCult-B™ basal medium supplemented with Mouse EpiCult-B™ supplements, 5% FBS, 10 ng/mL EGF, 10 ng/mL bFGF, 4 m g/mL heparin, and 50 m g/mL gentamicin ( see Note 13 ).

3.4. Mouse Mammary Colony-Forming Assays

40325 Mouse Mammary Cell Culture

3. Label the bottom of the 60 mm dishes before cell seeding. Do not label the lids as these can get easily mixed up when using a large number of dishes.

4. Add a suitable number of dissociated single cells to 8 mL of complete Mouse EpiCult-B™ ( see Notes 14 and 15 ). Add 50,000 viable irradiated NIH 3T3 cells per mL of complete Mouse EpiCult-B™ media. Seed the cell suspension across two duplicate 60 mm cell culture dishes at 4 mL per dish.

5. Incubate the cell culture dishes in a hypoxic incubator (5% oxygen, 5% carbon dioxide, 37°C) for 7 days ( see Note 16 ).

6. After 7 days, remove the media and gently rinse the dishes with PBS. Completely remove the PBS and add 1 mL of acetone–meth-anol (1:1) per 60 mm dish for 30 s. Remove the acetone–methanol and allow the dish to air-dry. Add 1–2 mL of Giemsa stain (diluted 1:10 with distilled water) for 2–3 min. Remove the Giemsa stain and rinse the dishes twice with distilled water and air-dry.

7. Count the number of colonies per dish using a scoring grid, which can be made by taking a 100 mm dish and making verti-cal lines using a fi ne-tip pen. The space between the lines should be one fi eld-of-view under the microscope.

Epithelial colonies can be derived from either luminal or basal cells and typically have a “cobblestone” appearance, although the basal colonies are usually more dispersed than the compact luminal colo-nies. Distinguishing luminal and basal colonies is dif fi cult by mor-phology alone. Immuno fl uorescence staining for cytokeratins 14 (K14) and 18 (K18) allows basal and luminal cells, respectively, to be distinguished. Colonies derived from luminal cells express K18 only. Basal cells usually give rise to mixed colonies that contain both K18 + luminal and K14 + basal cells. Other markers for luminal cells include MUC1 and K8, whereas basal cells can also be identi fi ed by expression of K5, p63, and smooth muscle actin ( 2, 12– 14 ) ( see Fig. 3 ).

Stromal colonies have a dispersed and mesenchymal phenotype and can be easily distinguished from epithelial colonies ( see Fig. 4 ). These colonies should be excluded from colony counts if only epi-thelial progenitors are of interest.

The frequency of mammary repopulating units (MRUs) within a mammary cell preparation can be estimated ( see Note 17 ) by trans-planting the cells at limiting dilutions (e.g., a dose where at least one negative outcome will be obtained) into cleared mouse mam-mary fat pads ( 4– 7 ) . MRUs are the operational term for cells that have the ability to generate ductal-lobular outgrowths when trans-planted into a cleared mammary fat pad. Limiting dilution analysis is a low-resolution tool, and as a result the 95% con fi dence inter-vals generated with the MRU frequency estimates are large. This

3.5. Characterization of Mouse Mammary Epithelial Cell Colonies

3.6. Detection of Mouse Mammary Stem Cells

404 M. Prater et al.

con fi dence interval can be narrowed by transplanting cells at limit-ing numbers, that is, at that dose in which an MRU may or may not be in the transplant inoculum. For example, if the MRU fre-quency is 1 MRU for every 1,000 total cells, then it would be more ef fi cient to transplant cells at a dose of 1,000 cells/transplant, rather than doses that are signi fi cantly higher or lower than this. Initial pilot transplants using a broad range of transplant doses may need to be performed to determine the approximate MRU fre-quency. A second round of transplants can then be done at more re fi ned doses to generate accurate MRU estimates. It is our experi-ence that different donor mouse strains have different MRU fre-quencies (e.g., 1 MRU in 100 total mammary cells isolated from FVB mice, and 1 MRU in 1,300 total mammary cells isolated from C57Bl6 mice). A suggested protocol to identify MRUs among total or fl ow-sorted cell preparations is as follows ( see Note 18 ):

Fig. 3. Colonies derived from a fl ow sorted luminal cell ( a ) and a basal cell ( b ) stained for cytokeratin 18 ( red ), cytokeratin 14 ( green ) and DAPI ( purple ) by immuno fl uorescence. Bar = 100 m m .

Fig. 4. Stromal colonies ( a and b ) grown in Mouse EpiCult-B™ media. Bar = 500 m m.

40525 Mouse Mammary Cell Culture

1. Suspend cells in 65% PBS + 25% Growth Factor Reduced Matrigel + 10% trypan blue solution (0.4%) at an appropriate concentration such that 10 m L contains the desired cell dose. Keep the cells on ice.

2. Clear the endogenous mammary gland epithelium from the inguinal (number 4) mammary glands of a 21 day-old female recipient mouse ( see Note 19 ).

3. Using a 25 m L Hamilton syringe and a 22 Gauge needle, inject 10 m L of cell suspension into each cleared fat pad ( see Note 20 ).

4. Mate the mice 3 weeks after surgery and remove the fat pads when the mice are visibly pregnant.

5. Fix the fat pads in 5 mL of 6 parts absolute ethanol, 3 parts chloroform and 1 part glacial acetic acid (Carnoy’s fi xative) overnight at room temperature.

6. Remove the Carnoy’s fi xative and wash in 5 mL of 70% ethanol for 15 min at room temperature.

7. Slowly add 5 mL of distilled water to the 70% ethanol to grad-ually reduce the concentration of ethanol and then wash the glands once in distilled water for 5 min. Remove the distilled water.

8. Make up carmine stain: 1 g carmine mixed with 2.5 g alumi-num potassium sulfate in 500 mL distilled water. Boil for 20 min. Filter through Whatman fi lter paper to remove any precipitates. Store at 4°C until color begins to fade.

9. Add 5 mL of carmine stain and leave overnight at room temperature.

10. Remove the carmine stain and wash sequentially in 70, 95, and 100% ethanol for 15 min each. Immerse in two sequential baths of xylene for 15 min each and mount with Permount Mounting Medium.

11. A positive engraftment must originate from within the fat pad ( see Note 21 ) and must contain ducts and lobules. Note how much of the fat pad has been fi lled by the engrafted epithelium.

12. To assess the self-renewal capacity of an MRU, the primary engraftment should be dissociated as described above ( see Subheading 3.1 ) and cells should be transplanted at limiting dilutions into cleared fat pads of secondary recipients.

1. It is not necessary to remove the lymph nodes from the mam-mary gland since these cells will be excluded during cell sort-ing, nor is it necessary to mince the glands prior to enzymatic dissociation.

4. Notes

406 M. Prater et al.

2. Keep all buffers cold and keep cells on ice to minimize cell clumping.

3. Cells can be evenly distributed among the different staining FACS™ tubes when doing fl ow cytometric analysis. However, when sorting cells rather than just analyzing, it is appropriate to minimize the number of cells in the control tubes since a large number of cells are not required to be analyzed for these controls; most of the cells from the original sample can be placed into the tube which contains the cells that will be sorted. This will minimize cell wastage.

4. The use of an anti-EpCAM antibody instead of an anti-CD24 antibody is recommended since the EpCAM/CD49f antibody combination permits better resolution of the luminal and basal cell subpopulations for some strains of mice (e.g., C57 Bl6).

5. In some cases it may be desirable to deplete the contaminating hematopoietic, stromal and endothelial cells using an immuno-magnetic approach (e.g., Mouse Epithelial Enrichment Kit from StemCell Technologies catalogue number 19758). This will free a channel on the fl ow cytometer and permits an addi-tional fl uorochrome to be used.

6. The inclusion of an anti-BP1 antibody in the lineage depletion cocktail results in depletion of a large proportion of stromal cells. If stromal cells are the cells of interest, it may be desirable to remove the BP1 antibody from the lineage depletion cocktail.

7. A thorough discussion of the fl ow cytometric analysis of cells derived from mouse mammary tissue is presented in ref. 15 .

8. Events with very high SSC have high levels of auto fl uorescence when analyzed on the channel used to detect Alexa Fluor 647; as a result it is best to gate out these events.

9. The luminal subpopulation can be further subdivided into pro-genitor and non-progenitor cell subpopulations by the expres-sion of CD61 ( see ref. 16 ) .

10. Approximately 70–80% of all MRUs are localized in the bright-est 20% of the EpCAM + cells within the basal cell population (e.g., in the red circle outlined in Fig. 2e ). Their frequency in this gate when analyzing C57 Bl6 mice is approximately 1 MRU in every 50 sorted cells.

11. The actual number of cells collected by FACS™ often does not mirror the number of events the machine states that it has sorted. It is recommended that a test sort is performed at the beginning of every sorting session. To do this, collect 100,000 DAPI − events and then count the number of viable cells in this cell preparation using trypan blue and a hemocytometer to determine the actual cell yield, and use this to calculate a sort

40725 Mouse Mammary Cell Culture

correction factor. Adjust all subsequent cell-sorting counts by this correction factor.

12. Do not allow cultures of NIH 3T3 mouse embryonic fi broblasts to become con fl uent or they will not support clonal growth of mammary epithelial cells. Over time, the NIH 3T3 cells may lose their ability to support clonogenic growth of the mam-mary cells. In such cases new stocks of feeder cells should be generated from early passage master stocks that are preserved in liquid nitrogen.

13. Add gentamicin to culture media if the cells to be cultured have been sorted by fl ow cytometry under non-sterile condi-tions. Do not use penicillin–streptomycin because this may in fl uence the number of epithelial colonies generated in vitro.

14. In our experience, the vast majority of progenitors that grow in Mouse EpiCult-B™ are progenitors that have a luminal-like phenotype and generate luminal-restricted progeny, although a small proportion (~1–3%) of progenitors are localized to the MRU gate and generate mixed lineage (luminal and basal) colonies in vitro.

Fig. 5. Mouse mammary epithelial colonies grown in Mouse-EpiCult-B™ in atmospheric oxygen ( a and b ) versus 5% oxygen ( c and d ). Bar = 500 m m .

408 M. Prater et al.

15. The number of cells seeded should be titrated the fi rst time around. The colony forming cell (Ma-CFC) frequency in unsorted mammary epithelial cells is approximately 3%. Choose a seeding density that results in 50–100 colonies because at this density individual colonies can be easily distinguished.

16. Although the numbers of epithelial colonies generated in hypoxic and normoxic conditions is equivalent, colonies gen-erated in hypoxic conditions are larger than those generated in normoxic conditions ( see Fig. 5 ).

17. The MRU frequency can be estimated using the L-Calc com-puter statistical program. The program can be downloaded for free from www.stemcell.com . The program is compatible with PCs only. Alternatively, the more robust ELDA computer sta-tistical program can be downloaded from http://bioinf.wehi.edu.au/software/elda and is compatible with both Macs and PCs.

18. There is a certain cell toxicity associated with antibody expo-sure and fl ow sorting among mouse mammary MRUs and Ma-CFCs. We have estimated this toxicity level to be approxi-mately 50%.

19. The endogenous mammary gland epithelium will not have grown past the lymph node in 21-day-old female mice. Remove all endogenous tissue that is lateral to the lymph node. A detailed protocol on mammary fat pad clearing can be found in ref. 17 .

20. Placing a small piece of Te fl on (plumber’s) tape around the end of the Hamilton syringe creates a better seal with the nee-dle. Also remove the plunger from the syringe and dip it into mineral oil. This thin coating of oil permits a better vacuum seal to be made between the barrel and the syringe.

21. If the endogenous mammary epithelium was not cleared prop-erly, false positive engraftments can occur. If the engraftment does not originate from within the fat pad but instead enters from the edges, then exclude it from the results. To avoid this problem, genetically tagged (e.g., eGFP) cells could be transplanted.

Acknowledgments

MP, MS, and JS are funded by Cancer Research UK, The University of Cambridge, and Hutchison Whampoa Limited. MP and JS are also funded by the Breast Cancer Campaign. CJW is funded by the Biotechnology and Biological Sciences Research Council, the Medical Research Council (UK), and the Breast Cancer Campaign.

40925 Mouse Mammary Cell Culture

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