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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. doi: 10.17917/YTHD-KF51</p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/nciprotocols/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-nciprotocols-lrg.png" alt="Cover of National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK604939_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK604939_dtls__"><div>Bethesda (MD): <a href="https://www.cancer.gov/nano/research/ncl" ref="pagearea=page-banner&targetsite=external&targetcat=link&targettype=publisher">National Cancer Institute (US)</a>; 2005 May 1-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/nciprotocols/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/nciprotocols/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/nciprotocols/ncipr42/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/nciprotocols/ncipr38/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK604939_"><span class="label">NCL Method ITA-3</span><span class="title" itemprop="name">Mouse Granulocyte-Macrophage Colony-Forming Unit Assay</span></h1><div class="subtitle whole_rhythm">Version 4</div><p class="contrib-group"><span itemprop="author">Barry W. Neun</span>, <span itemprop="author">Edward Cedrone</span>, and <span itemprop="author">Marina A. Dobrovolskaia</span>.</p><a data-jig="ncbitoggler" href="#__NBK604939_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK604939_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Barry W. Neun</span>, <span itemprop="author">Edward Cedrone</span>, and <span itemprop="author">Marina A. Dobrovolskaia</span><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup><sup>1</sup>.</p><h4>Contact</h4><div class="affiliation"><sup>1</sup> <span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.liam@aniram" class="oemail">vog.hin.liam@aniram</a></div><div class="affiliation"><sup>1</sup> Nanotechnology Characterization Lab, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, MD 21702</div><div><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author.</div></div><p class="small">Published: <span itemprop="datePublished">May 2020</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="ncipr39.s1"><h2 id="_ncipr39_s1_">1. Introduction</h2><p>Myelosuppression is a common dose limiting toxicity of cytotoxic oncology drugs. Nanoparticles may distribute to bone marrow and/or release drug that is delivered to bone marrow. Therefore, understanding potential toxicity of nanoparticles or drugs which nanoparticles carry is an important step in preclinical safety evaluation.</p><p>Hematopoietic stem cells of bone marrow (BM) proliferate and differentiate to form discrete cell clusters or colonies. This document describes a protocol for quantitative analysis of granulocyte-macrophage (GM) colony-forming units (CFU), employing murine BM. This protocol can be used for both <i>in vitro</i> and <i>ex vivo</i> analyses. The <i>in vitro</i> protocol involves isolation of bone marrow cells from healthy animals, followed by treatment <i>in vitro</i> with nanoparticle formulations. In the <i>ex vivo</i> version, the bone marrow is isolated from animals injected with the nanoparticle formulation. The <i>in vitro</i> protocol does not account for nanoparticle biodistribution; however, in cases when dose information is not available and nanoparticle formulation is in the early phase of development, the <i>in vitro</i> protocol allows for rapid screening of potentially toxic nanoparticle formulations. The <i>in vitro</i> protocol can also give a quick estimation of the myelosuppressive potential of a cytotoxic oncology drug bound to a nanoparticle surface in comparison to a traditional formulation of the same cytotoxic oncology drug (for an example, see <a class="figpopup" href="/books/NBK604939/figure/ncipr39.fig1/?report=objectonly" target="object" rid-figpopup="figncipr39fig1" rid-ob="figobncipr39fig1">Figure 1</a>).</p></div><div id="ncipr39.s2"><h2 id="_ncipr39_s2_">2. Principles</h2><p>The protocol described here details the <i>in vitro</i> CFU-GM study. BM cells are isolated from 8-12 week old mice and cultured in methylcellulose-based medium supplemented with cytokines (mSCF, mIL-3 and hIL-6) and are either untreated (baseline) or treated with nanoparticles (test). The cytokines used in the MethoCult media promote formation of granulocyte and macrophage (CFU-GM) colonies. After a twelve days incubation period of the BM cells, the number of colonies is quantified in both the baseline and test samples. Comparison between the baseline and test samples allows the identification of test materials which can inhibit CFU-GM formation. The basic protocol for BM isolation and culture was adopted from technical manual # 28405 developed by StemCell Technologies, Inc [<a class="bk_pop" href="#ncipr39.ref1">1</a>–<a class="bk_pop" href="#ncipr39.ref2">2</a>]. If the <i>ex vivo</i> study is required, <a href="#ncipr39.s4">steps 4</a>, <a href="#ncipr39.lt40">7.9</a> and <a href="#ncipr39.lt49">8.5</a> are modified (please refer to <a href="#ncipr39.s4">sections 4</a>, <a href="#ncipr39.s7">7</a> and <a href="#ncipr39.s8">8</a> for details).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figncipr39fig1" co-legend-rid="figlgndncipr39fig1"><a href="/books/NBK604939/figure/ncipr39.fig1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figncipr39fig1" rid-ob="figobncipr39fig1"><img class="small-thumb" src="/books/NBK604939/bin/ncipr39f1.gif" src-large="/books/NBK604939/bin/ncipr39f1.jpg" alt="An example excel graph showing varying CFU-GM numbers for different concentration samples." /></a><div class="icnblk_cntnt" id="figlgndncipr39fig1"><h4 id="ncipr39.fig1"><a href="/books/NBK604939/figure/ncipr39.fig1/?report=objectonly" target="object" rid-ob="figobncipr39fig1">Figure 1</a></h4><p class="float-caption no_bottom_margin"><i>In vitro</i> comparison of traditional and nano-formulated cytotoxic oncology drug in CFU-GM assay. Bone marrow cells were treated <i>in vitro</i> with nanoparticle-bound cytotoxic oncology drug or with traditional formulation of the same cytotoxic oncology drug. <a href="/books/NBK604939/figure/ncipr39.fig1/?report=objectonly" target="object" rid-ob="figobncipr39fig1">(more...)</a></p></div></div></div><div id="ncipr39.s3"><h2 id="_ncipr39_s3_">3. Reagents, Materials, and Equipment</h2><blockquote><p><i>Note: The NCL does not endorse any of the suppliers listed below; these reagents were used in the development of the protocol and their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted. Please note that suppliers may undergo a name change due to a variety of factors. Brands and part numbers typically remain consistent but may also change over time</i>.</p></blockquote><dl id="ncipr39.l1" class="temp-labeled-list"><dt>3.1.</dt><dd id="ncipr39.lt1"><p class="no_top_margin">Reagents
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<dl id="ncipr39.l2" class="temp-labeled-list"><dt>3.1.1.</dt><dd id="ncipr39.lt2"><p class="no_top_margin">MethoCult medium (Stem Cell Technologies, Inc., 03534)</p></dd><dt>3.1.2.</dt><dd id="ncipr39.lt3"><p class="no_top_margin">Fetal Bovine Serum prescreened for hematopoietic stem cells (Stem Cell Technologies, Inc., 06200)</p></dd><dt>3.1.3.</dt><dd id="ncipr39.lt4"><p class="no_top_margin">Iscove’s MDM (IMDM) with 2% FBS (Stem Cell Technologies, Inc., 07700)</p></dd><dt>3.1.4.</dt><dd id="ncipr39.lt5"><p class="no_top_margin">Sterile distilled water</p></dd><dt>3.1.5.</dt><dd id="ncipr39.lt6"><p class="no_top_margin">Cisplatin (positive control) (Sigma, P4394)</p></dd><dt>3.1.6.</dt><dd id="ncipr39.lt7"><p class="no_top_margin">Sterile Ca<sup>2+</sup>/Mg<sup>2+</sup>-free PBS (GE Life Sciences, HyClone, SH30256.01)</p></dd><dt>3.1.7.</dt><dd id="ncipr39.lt8"><p class="no_top_margin">3% Acetic Acid with Methylene Blue (Stem Cell Technologies, Inc., 07060)</p></dd></dl></p></dd><dt>3.2.</dt><dd id="ncipr39.lt9"><p class="no_top_margin">Materials
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<dl id="ncipr39.l3" class="temp-labeled-list"><dt>3.2.1.</dt><dd id="ncipr39.lt10"><p class="no_top_margin">Pipettes covering the range of 0.05 to 10 mL</p></dd><dt>3.2.2.</dt><dd id="ncipr39.lt11"><p class="no_top_margin">Prescreened 35 mm culture dishes (Stem Cell Technologies, Inc., 27100)</p></dd><dt>3.2.3.</dt><dd id="ncipr39.lt12"><p class="no_top_margin">Blunt-end, 16-gauge needles (Stem Cell Technologies, Inc., 28110)</p></dd><dt>3.2.4.</dt><dd id="ncipr39.lt13"><p class="no_top_margin">100 mm Petri dishes</p></dd><dt>3.2.5.</dt><dd id="ncipr39.lt14"><p class="no_top_margin">Plastic beakers</p></dd><dt>3.2.6.</dt><dd id="ncipr39.lt15"><p class="no_top_margin">Polypropylene tubes, 5 and 15 mL</p></dd><dt>3.2.7.</dt><dd id="ncipr39.lt16"><p class="no_top_margin">Scissors for tissue dissection</p></dd><dt>3.2.8.</dt><dd id="ncipr39.lt17"><p class="no_top_margin">Forceps</p></dd></dl></p></dd><dt>3.3.</dt><dd id="ncipr39.lt18"><p class="no_top_margin">Equipment
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<dl id="ncipr39.l4" class="temp-labeled-list"><dt>3.3.1.</dt><dd id="ncipr39.lt19"><p class="no_top_margin">Centrifuge</p></dd><dt>3.3.2.</dt><dd id="ncipr39.lt20"><p class="no_top_margin">Refrigerator, 2-8ºC</p></dd><dt>3.3.3.</dt><dd id="ncipr39.lt21"><p class="no_top_margin">Freezer, −20ºC</p></dd><dt>3.3.4.</dt><dd id="ncipr39.lt22"><p class="no_top_margin">Cell culture incubator with 5% CO<sub>2</sub> and 95% humidity</p></dd><dt>3.3.5.</dt><dd id="ncipr39.lt23"><p class="no_top_margin">CO<sub>2</sub> euthanasia box, or appropriate equipment approved by your organization</p></dd><dt>3.3.6.</dt><dd id="ncipr39.lt24"><p class="no_top_margin">Biohazard safety cabinet approved for level II handling of biological material</p></dd><dt>3.3.7.</dt><dd id="ncipr39.lt25"><p class="no_top_margin">Inverted microscope</p></dd><dt>3.3.8.</dt><dd id="ncipr39.lt26"><p class="no_top_margin">Vortex</p></dd><dt>3.3.9.</dt><dd id="ncipr39.lt27"><p class="no_top_margin">Hemocytometer</p></dd></dl></p></dd></dl></div><div id="ncipr39.s4"><h2 id="_ncipr39_s4_">4. Animals</h2><p>This protocol utilizes 8-12 week old, C56BL6 males or females. Use of pooled cells derived from at least two (<a class="bk_pop" href="#ncipr39.ref2">2</a>) animals is highly recommended. The exception is when the experiment is conducted to support an in vivo study in which the animals have been injected with test nanoparticles. In this case, process each animal separately.</p><p>NCI-Frederick is accredited by AAALAC International and follows the Public Health Service <i>Policy for the Care and Use of Laboratory Animals</i> (Health Research Extension Act of 1985, Public Law 99-158, 1986). Animal care is provided in accordance with the procedures outlined in the <i>Guide for Care and Use of Laboratory Animals</i> (National Research Council, 1996; National Academy Press, Washington, D.C.). All animal protocols are approved by the NCI-Fredrick institutional Animal Care and Use Committee.</p><p>If this procedure is conducted outside NCI at Frederick facilities, one has to ensure that animal work is supported by approved protocols.</p></div><div id="ncipr39.s5"><h2 id="_ncipr39_s5_">5. Reagent and Control Preparation</h2><dl id="ncipr39.l5" class="temp-labeled-list"><dt>5.1.</dt><dd id="ncipr39.lt28"><p class="no_top_margin">
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<u>MethoCult Medium</u>
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</p><p>The MethoCult medium is supplied in 100 mL size batches. This medium can be thawed and used fresh or aliquoted into single 3 mL volumes in 15 mL conical tubes. It is recommended by the manufacturer that the medium be thawed at room temperature, or in a refrigerator overnight, vortexed to mix well, then kept at room temperature for approximately 5 min to allow air bubbles to dissipate. Use 16-gauge blunt-end needles to aliquot the MethoCult medium. Store the aliquots at a nominal temperature of −20ºC. Before the test, thaw the required number of aliquots at room temperature for approximately 20 min and keep on ice prior to use. Alternatively, they can be thawed on ice or in refrigerator. Avoid repeated freeze/thaws.</p></dd><dt>5.2.</dt><dd id="ncipr39.lt29"><p class="no_top_margin">
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<u>50 mM Cisplatin (Positive Control)</u>
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</p><p>Cisplatin is supplied in a lyophilized form. Reconstitute the lyophilized powder by adding an appropriate amount of DMSO to make a stock solution with nominal concentration of 50 mM. Prepare small aliquots and store at a nominal temperature of −20ºC or lower. Prior to use in the assay, thaw an aliquot of the stock solution at room temperature and dilute in IMDM supplemented with 2% FBS to bring the concentration to 1.1 mM. 150 µL of this intermediate solution is then added to 3 mL of MethoCult culture medium. The final concentration of cisplatin in the positive control sample is 50 µM.
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<blockquote><p><i>Note: This control is not required if the ex vivo study is conducted, because the ex vivo study has its own negative and positive controls</i>.</p></blockquote></p></dd><dt>5.3.</dt><dd id="ncipr39.lt30"><p class="no_top_margin">
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<u>Negative Control (PBS)</u>
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</p><p>Sterile Ca<sup>2+</sup>/Mg<sup>2+</sup>-free PBS is used as a negative control. Store at room temperature for up to 6 months. Dilute this sample the same way you dilute the test nanomaterials.</p></dd><dt>5.4.</dt><dd id="ncipr39.lt31"><p class="no_top_margin">
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<u>Vehicle Control (relevant to each given nanoparticle)</u>
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</p><p>When nanoparticles are not formulated in saline or PBS, the vehicle should be tested to estimate the effect of excipients on the bone marrow precursors. This control is specific to each given nanoparticle sample. Vehicle control should match formulation buffer of the test nanomaterial by both the composition and concentration. Dilute this sample the same way you dilute the test nanomaterials. If the vehicle is PBS this control can be skipped.</p></dd></dl></div><div id="ncipr39.s6"><h2 id="_ncipr39_s6_">6. Preparation of Study Samples</h2><p>This assay requires 0.6 mL of nanoparticle solution at 22X the highest test concentration. The media for the stock material and subsequent dilutions should be determined for each nanoformulation. Ideally, the nanoparticle should be dissolved/resuspended in IMDM medium. However, often this is not possible due to the limited concentration of the nanoparticle stock solution. If this is the case use nanoparticles directly from stock and prepare all dilutions in the same buffer as used for nanoparticle storage (e.g. if the stock is in PBS, use it directly and prepare dilutions in PBS; if the stock is in a vehicle different than PBS, use directly from stock and use that vehicle to prepare all dilutions). The concentration is selected based on the plasma concentration of the nanoparticle at the intended therapeutic dose. For the purpose of this protocol this concentration is called “theoretical plasma concentration”. Considerations for estimating theoretical plasma concentration were reviewed elsewhere [<a class="bk_pop" href="#ncipr39.ref3">3</a>] and are summarized in <a href="/books/NBK604939/box/ncipr39.box6/?report=objectonly" target="object" rid-ob="figobncipr39box6">Box 1</a> below.</p><p>The assay will evaluate 4 concentrations: 10X (or when feasible 100X, 30X or 5X) of the theoretical plasma concentration, theoretical plasma concentration and two 1:5 serial dilutions of the theoretical plasma concentration. When the intended therapeutic concentration is unknown, the highest final concentration is 1 mg/mL or the highest reasonably achievable concentration.</p><p>For example, if the final theoretical plasma concentration to be tested is 0.2 mg/mL, then a stock of 44 mg/mL will be prepared and diluted 10-fold (4.4 mg/mL), followed by two 1:5 serial dilutions (0.88 and 0.18 mg/mL). When 150 μL of each of these samples is added to the test tube and mixed with 3 mL of MethoCult medium and 0.15 mL of cell suspension, the final nanoparticle concentrations tested in the assay are: 2.0, 0.2, 0.04 and 0.008 mg/mL. Three 150 μL replicates are tested per each sample concentration.</p><div id="ncipr39.box6" class="box"><h3><span class="label">Box 1</span><span class="title">Example Calculation to Determine Nanoparticle Concentration for In Vitro Tests</span></h3><p>In this example, we assume a mouse dose of 123 mg/kg. Therefore, the scaled equivalent human dose would be:
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr39.deq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr39.eq1" display="block"><mi>H</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi><mo>=</mo><mfrac><mrow><mi>m</mi><mi>o</mi><mi>u</mi><mi>s</mi><mi>e</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi></mrow><mrow><mn>12.3</mn></mrow></mfrac><mo>=</mo><mfrac><mrow><mn>123</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></mrow><mrow><mn>12.3</mn></mrow></mfrac><mo>=</mo><mn>10</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div>
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Blood volume constitutes approximately 8% of the body weight. Therefore, an average human of 70 kg body weight has approximately 5.6 L of blood. Assuming all the nanoparticle injected goes into the systemic circulation, this provides a rough approximation of the potential maximum nanoparticle concentration in blood, which is used as the in vitro test concentration.</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr39.deq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">
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<math id="ncipr39.eq2" display="block"><mi>i</mi><mi>n</mi><mtext> </mtext><mi>v</mi><mi>i</mi><mi>t</mi><mi>r</mi><mi>o</mi><mtext> </mtext><mi>c</mi><mi>o</mi><mi>n</mi><mi>c</mi><mi>e</mi><mi>n</mi><mi>t</mi><mi>r</mi><mi>a</mi><mi>t</mi><mi>i</mi><mi>o</mi><msub><mi>n</mi><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>m</mi><mi>a</mi><mi>t</mi><mi>r</mi><mi>i</mi><mi>x</mi></mrow></msub><mo>=</mo><mfrac><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi></mrow><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>b</mi><mi>l</mi><mi>o</mi><mi>o</mi><mi>d</mi><mtext> </mtext><mi>v</mi><mi>o</mi><mi>l</mi><mi>u</mi><mi>m</mi><mi>e</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><mn>70</mn><mtext> </mtext><mi>k</mi><mi>g</mi><mtext> </mtext><mo>∗</mo><mtext> </mtext><mn>10</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></mrow><mrow><mn>5.6</mn><mtext> </mtext><mi>L</mi></mrow></mfrac><mo>=</mo><mn>0.125</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>m</mi><mi>L</mi></math>
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</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div></div></div><div id="ncipr39.s7"><h2 id="_ncipr39_s7_">7. Isolation and Counting of Bone Marrow Cells</h2><dl id="ncipr39.l6" class="temp-labeled-list"><dt>7.1.</dt><dd id="ncipr39.lt32"><p class="no_top_margin">Position euthanized mouse on its back and rinse fur thoroughly with 70% alcohol. (Euthanize animals according to the protocol approved by your institution.)</p></dd><dt>7.2.</dt><dd id="ncipr39.lt33"><p class="no_top_margin">Cut a slit in the fur just below the rib cage without cutting the peritoneal membrane.</p></dd><dt>7.3.</dt><dd id="ncipr39.lt34"><p class="no_top_margin">Firmly grasp skin and pull back to expose hind limbs.</p></dd><dt>7.4.</dt><dd id="ncipr39.lt35"><p class="no_top_margin">Using sterile sharp dissecting scissors cut the knee joint in the center. Cut through ligaments and excess tissue.</p></dd><dt>7.5.</dt><dd id="ncipr39.lt36"><p class="no_top_margin">Grasp the femur with forceps and cut the femur near the hip joint.</p></dd><dt>7.6.</dt><dd id="ncipr39.lt37"><p class="no_top_margin">Free the tibia by cutting near the ankle joint.</p></dd><dt>7.7.</dt><dd id="ncipr39.lt38"><p class="no_top_margin">Trim the ends of the long bones to expose the interior marrow shaft. Put bones in a sterile Petri dish, or in sterile culture medium, and place on ice. Bones can be collected from multiple animals.</p></dd><dt>7.8.</dt><dd id="ncipr39.lt39"><p class="no_top_margin">Using a 3cc syringe with a 21 or 22 gauge needle, draw up to 1-3 mL of cold Iscove’s MDM supplemented with 2% FBS.</p></dd><dt>7.9.</dt><dd id="ncipr39.lt40"><p class="no_top_margin">Insert the bevel of the needle into marrow shaft and flush marrow into a 15 mL tube. Repeat this procedure for all bones. The same medium can be used to isolate marrow from 1-3 animals. Once all the marrow has been expelled the bone should appear white. The exception is when this protocol is used to isolate BM from individual animals from an in vivo study. In this case BM from each animal is isolated into 3mL of medium in a separate tube.
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<blockquote><p><i><b>Note</b>: Typically, 3mL of medium is enough to collect BM from 6 femurs (i.e. from 3 animals); however, when more animals are used, it is better to collect cells into a higher volume of media (e.g. 3 mL per each 6 femurs). Additional 1-3 mL of fresh medium may be used to flush the BM shaft after first flush. If this is the case the total volume of the cell suspension will be more than 3 mL and one may need to concentrate the cells before proceeding with the assay. Concentration by centrifugation is described in the note to <a href="#ncipr39.lt43">section 7.12</a> below</i>.</p></blockquote></p></dd><dt>7.10.</dt><dd id="ncipr39.lt41"><p class="no_top_margin">Keeping the needle below the medium surface, gently draw medium with cells up and down using a 3cc syringe with a 21-gauge needle, 3-4 times, to make a single cell suspension.</p></dd><dt>7.11.</dt><dd id="ncipr39.lt42"><p class="no_top_margin">Keep cells in medium, on ice until use.</p></dd><dt>7.12.</dt><dd id="ncipr39.lt43"><p class="no_top_margin">Perform a nucleated cell count. To do so, first dilute the cells 1:100 with 3% acetic acid with methylene blue (e.g., 10 µL cells + 990 µL 3% acetic acid/methylene blue). Then, use either hemocytometer or automatic cell counter to obtain counts. An average cell count is expected to be 1 × 10<sup>7</sup> to 2 × 10<sup>7</sup> from the femur and 0.6 × 10<sup>7</sup> to 1 × 10<sup>7</sup> from the tibia.
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<blockquote><p><i><b>Note: A)</b> If cells were extracted into larger volume of media, one may concentrate the cells before counting. To do so spin down isolated cells at 700xg. The spinning time is estimated such as to spin 5 minutes for each 3 mL (e.g., if the total volume to be spun is 3 mL, spinning time is 5 min; if the volume of the cell suspension in the tube is 6mL, spinning time is 10 min etc.)</i></p><p><i><b>B)</b> While other dyes (e.g., AOPI or trypan blue) can be used to determine cell count and viability, especially when automated cell counters are utilized, manual counting of bone marrow cells is more accurate with the 3% acetic acid methylene blue; this solution eliminates anucleated cells from count. The nuclei of pluripotent stem cells are stained light blue</i>.</p></blockquote></p></dd><dt>7.13.</dt><dd id="ncipr39.lt44"><p class="no_top_margin">If cell viability (at least 90%) and count are acceptable, proceed to the next step.</p></dd></dl></div><div id="ncipr39.s8"><h2 id="_ncipr39_s8_">8. Experimental Procedure</h2><dl id="ncipr39.l7" class="temp-labeled-list"><dt>8.1.</dt><dd id="ncipr39.lt45"><p class="no_top_margin">Label lids of 35 mm culture dishes at the edge using a permanent, fine tip, felt marker.</p></dd><dt>8.2.</dt><dd id="ncipr39.lt46"><p class="no_top_margin">Thaw MethoCult medium at room temperature or in refrigerator overnight.</p></dd><dt>8.3.</dt><dd id="ncipr39.lt47"><p class="no_top_margin">Vortex tubes to ensure all components are thoroughly mixed.</p></dd><dt>8.4.</dt><dd id="ncipr39.lt48"><p class="no_top_margin">Dilute the isolated cells (<a href="#ncipr39.s7">section 7</a>) with Iscove’s medium supplemented with 2% FBS to 4 × 10<sup>5</sup> cells/mL.</p></dd><dt>8.5.</dt><dd id="ncipr39.lt49"><p class="no_top_margin">Add 150 µL of cell suspension and 150 µL of either Iscove’s medium with 2% FBS (baseline), negative control, positive control, test sample or vehicle control to 3 mL of MethoCult medium. Test three replicates (n=3) for each test concentration.
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<blockquote><p><i><b>Note</b>: If the ex vivo study is conducted, add 150</i> µ<i>L of cell suspension from individual animals and 150</i> µ<i>L of Iscove’s medium with 2% FBS to 3 mL of Methocult medium. No addition of PBS, nanoparticles or cisplatin is required because animals were previously injected with various formulations representing vehicle control, nanoparticle treatment and/or traditional cytotoxic oncology drug formulation</i>.</p></blockquote></p></dd><dt>8.6.</dt><dd id="ncipr39.lt50"><p class="no_top_margin">Vortex the tubes to ensure all cells and medium components are mixed thoroughly.</p></dd><dt>8.7.</dt><dd id="ncipr39.lt51"><p class="no_top_margin">Let the tubes stand for 5 min to allow bubbles to dissipate.</p></dd><dt>8.8.</dt><dd id="ncipr39.lt52"><p class="no_top_margin">Attach a 16-gauge blunt ended needle to a 3cc syringe; place the needle below the surface of the solution containing Methocult media with cells in it from <a href="#ncipr39.lt49">steps 8.5</a>-<a href="#ncipr39.lt51">8.7</a> and draw up approximately 1 mL. Gently depress the plunger and expel medium completely. Repeat until no air space is visible.</p></dd><dt>8.9.</dt><dd id="ncipr39.lt53"><p class="no_top_margin">Draw up MethoCult medium with cells into the syringe and dispense 1.1 mL per 35 mm dish. All samples are tested in duplicate (n=2) (i.e., prepare two 35 mm dishes for each sample tested).</p></dd><dt>8.10.</dt><dd id="ncipr39.lt54"><p class="no_top_margin">Distribute the medium evenly by gently tilting and rotating each dish.</p></dd><dt>8.11.</dt><dd id="ncipr39.lt55"><p class="no_top_margin">Place the two covered dishes with cells and one uncovered dish filled with 3 mL of sterile water, into a 100 mm Petri dish.</p></dd><dt>8.12.</dt><dd id="ncipr39.lt56"><p class="no_top_margin">Place cultures in an incubator maintained at 37ºC, 5% CO<sub>2</sub> and 95% humidity.</p></dd><dt>8.13.</dt><dd id="ncipr39.lt57"><p class="no_top_margin">Incubate for 12 days. On the 12<sup>th</sup> day, remove dishes from incubator, identify and count colonies as described below. Representative values of CFU-GM for C57BL6 mice at 8-12 weeks of age is 64 ± 16.</p></dd></dl></div><div id="ncipr39.s9"><h2 id="_ncipr39_s9_">9. Description of CFU-GM</h2><p>This classification includes CFU-granulocyte (CFU-G), CFU-macrophage (CFU-M) and CFU-granulocyte macrophage (CFU-GM). The colonies contain 30 to thousands of CFU-G, CFU-M or both cell types (CFU-GM). CFU-GM colonies often contain multiple clusters and appear as a dense core surrounded by cells. The monocytic lineage cells are large cells with an oval to round shape and appear to have a grainy or grey center. The granulocytic lineage cells are round, bright, and are much smaller and more uniform in size than macrophages. It is easy to see individual cells of a CFU-GM colony, especially in the periphery of the colony. See <a class="figpopup" href="/books/NBK604939/figure/ncipr39.fig2/?report=objectonly" target="object" rid-figpopup="figncipr39fig2" rid-ob="figobncipr39fig2">Figure 2</a> below for a depiction of CFU-GM, CFU-M, and CFU-G colonies.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figncipr39fig2" co-legend-rid="figlgndncipr39fig2"><a href="/books/NBK604939/figure/ncipr39.fig2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figncipr39fig2" rid-ob="figobncipr39fig2"><img class="small-thumb" src="/books/NBK604939/bin/ncipr39f2.gif" src-large="/books/NBK604939/bin/ncipr39f2.jpg" alt="Figure 2. Depiction of CFU-GM, CFU-M, and CFU-G Colonies." /></a><div class="icnblk_cntnt" id="figlgndncipr39fig2"><h4 id="ncipr39.fig2"><a href="/books/NBK604939/figure/ncipr39.fig2/?report=objectonly" target="object" rid-ob="figobncipr39fig2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Depiction of CFU-GM, CFU-M, and CFU-G Colonies. <i>A</i> and <i>B</i> are CFU-GM colonies. <i>C</i> and <i>D</i> are CFU-M colonies. <i>E</i> depicts a single CFU-G colony. <i>F</i> shows a few CFU-G colonies growing together. </p></div></div></div><div id="ncipr39.s10"><h2 id="_ncipr39_s10_">10. Calculations</h2><p>The following parameter should be calculated for each control and test sample:
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<dl id="ncipr39.l8" class="temp-labeled-list"><dt>10.1.</dt><dd id="ncipr39.lt58"><p class="no_top_margin"><u>Percent Coefficient of Variation</u>
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||
<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr39.deq3"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr39.eq3" display="block"><mrow><mi>%</mi><mi>C</mi><mi>V</mi><mo>=</mo><mfrac><mrow><mi>s</mi><mi>t</mi><mi>a</mi><mi>n</mi><mi>d</mi><mi>a</mi><mi>r</mi><mi>d</mi><mtext> </mtext><mi>d</mi><mi>e</mi><mi>v</mi><mi>i</mi><mi>a</mi><mi>t</mi><mi>i</mi><mi>o</mi><mi>n</mi></mrow><mrow><mi>m</mi><mi>e</mi><mi>a</mi><mi>n</mi></mrow></mfrac><mo>∗</mo><mn>100</mn><mi>%</mi></mrow></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div></p></dd><dt>10.2.</dt><dd id="ncipr39.lt59"><p class="no_top_margin"><u>Percent CFU Inhibition</u></p><p>When analysis is performed to compare traditional and nanoformulated cytotoxic oncology drugs, calculating percent CFU inhibition may be helpful. Percent CFU inhibition is also helpful to compare data between experiments and between different strains if mouse strain other than C57BL6 is needed to address project specific needs.
|
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr39.deq4"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr39.eq4" display="block"><mrow><mi>%</mi><mtext> </mtext><mi>C</mi><mi>F</mi><mi>U</mi><mtext> </mtext><mi>I</mi><mi>n</mi><mi>h</mi><mi>i</mi><mi>b</mi><mi>i</mi><mi>t</mi><mi>i</mi><mi>o</mi><mi>n</mi><mo>=</mo><mfrac><mrow><mo stretchy="false">(</mo><mi>B</mi><mi>a</mi><mi>s</mi><mi>e</mi><mi>l</mi><mi>i</mi><mi>n</mi><mi>e</mi><mtext> </mtext><mi>C</mi><mi>F</mi><mi>U</mi><mtext>-</mtext><mi>G</mi><mi>M</mi><mtext> </mtext><mo>−</mo><mtext> </mtext><mi>T</mi><mi>e</mi><mi>s</mi><mi>t</mi><mtext> </mtext><mi>C</mi><mi>F</mi><mi>U</mi><mtext>-</mtext><mi>G</mi><mi>M</mi><mo stretchy="false">)</mo></mrow><mrow><mi>B</mi><mi>a</mi><mi>s</mi><mi>e</mi><mi>l</mi><mi>i</mi><mi>n</mi><mi>e</mi><mtext> </mtext><mi>C</mi><mi>F</mi><mi>U</mi><mtext> </mtext><mo>−</mo><mtext> </mtext><mi>G</mi><mi>M</mi></mrow></mfrac><mo>∗</mo><mn>100</mn><mi>%</mi></mrow></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div></p></dd></dl></p></div><div id="ncipr39.s11"><h2 id="_ncipr39_s11_">11. Acceptance Criteria</h2><dl id="ncipr39.l9" class="temp-labeled-list"><dt>11.1.</dt><dd id="ncipr39.lt60"><p class="no_top_margin">%CV for each control and test sample should less than 30%.</p></dd><dt>11.2.</dt><dd id="ncipr39.lt61"><p class="no_top_margin">If positive control or negative control fails to meet acceptance criterion described in 11.1, the assay should be repeated.</p></dd><dt>11.3.</dt><dd id="ncipr39.lt62"><p class="no_top_margin">Within the acceptable assay, if two of three replicates of unknown sample fail to meet acceptance criterion described in 11.1, this unknown sample should be re-analyzed.</p></dd></dl></div><div id="ncipr39.rl.r1"><h2 id="_ncipr39_rl_r1_">12. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ncipr39.ref1">Mouse Colony-Forming cell Assays Using MethoCult. Technical manual. StemCell Technologies Inc., cat # 28405.</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ncipr39.ref2">Dominique Pifat. Assay Validation. <a href="http://www.fda.gov/cber/summaries/120600bio10.ppt%20-%2005-24-2003" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">http://www<wbr style="display:inline-block"></wbr>.fda.gov/cber<wbr style="display:inline-block"></wbr>/summaries/120600bio10.ppt-05-24-2003</a></div></dd><dt>3.</dt><dd><div class="bk_ref" id="ncipr39.ref3">Dobrovolskaia
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MA, McNeil
|
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SE. Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines. J Control Release. 2013;172(2):456–66.
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[<a href="/pmc/articles/PMC5831149/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC5831149</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23742883" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23742883</span></a>]</div></dd></dl></div><div id="ncipr39.s12"><h2 id="_ncipr39_s12_">13. Abbreviations</h2><dl><dt id="ncipr39.abb_DL1_DI1">AAALAC</dt><dd><p>Association for Assessment and Accreditation of Laboratory Animal Care</p></dd><dt id="ncipr39.abb_DL1_DI2">API</dt><dd><p>active pharmaceutical ingredient</p></dd><dt id="ncipr39.abb_DL1_DI3">BM</dt><dd><p>bone marrow</p></dd><dt id="ncipr39.abb_DL1_DI4">CFU</dt><dd><p>colony forming units</p></dd><dt id="ncipr39.abb_DL1_DI5">CFU-G</dt><dd><p>colony forming unit-granulocyte</p></dd><dt id="ncipr39.abb_DL1_DI6">CFU-GM</dt><dd><p>colony forming unit-granulocyte macrophage</p></dd><dt id="ncipr39.abb_DL1_DI7">CFU-M</dt><dd><p>colony forming unit- macrophage</p></dd><dt id="ncipr39.abb_DL1_DI8">CV</dt><dd><p>coefficient of variation</p></dd><dt id="ncipr39.abb_DL1_DI9">FBS</dt><dd><p>fetal bovine serum</p></dd><dt id="ncipr39.abb_DL1_DI10">hIL-6</dt><dd><p>human interleukin-6</p></dd><dt id="ncipr39.abb_DL1_DI11">IMDM</dt><dd><p>Iscove’s modified Dulbecco’s medium</p></dd><dt id="ncipr39.abb_DL1_DI12">mIL-3</dt><dd><p>mouse interleukin-3</p></dd><dt id="ncipr39.abb_DL1_DI13">mSCF</dt><dd><p>mouse stem cell factor</p></dd><dt id="ncipr39.abb_DL1_DI14">PBS</dt><dd><p>phosphate buffered saline</p></dd><dt id="ncipr39.abb_DL1_DI15">SD</dt><dd><p>standard deviation</p></dd></dl></div><div><dl class="temp-labeled-list small"><dt></dt><dd><div><p class="no_top_margin"><p>This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.</p></p></div></dd><dt>*</dt><dd><div id="ncipr39.fn1"><p class="no_top_margin">Address correspondence to: <a href="mailto:dev@null" data-email="vog.hin.liam@aniram" class="oemail">vog.hin.liam@aniram</a></p></div></dd><dt></dt><dd><div><p class="no_top_margin"><div>
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<span class="mixed-citation" id="ncipr39.suggestedcitation">Neun BW, Cedrone E, Dobrovolskaia MA, NCL Method ITA-3: Mouse Granulocyte-Macrophage Colony-Forming Unit Assay. <a href="https://ncl.cancer.gov/resources/assay-cascade-protocols" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">https://www.cancer.gov/nano/research/ncl/protocols-capabilities</a> DOI: <a href="http://dx.crossref.org/10.17917/YTHD-KF51" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/YTHD-KF51</a></span>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div><div class="small"><span class="label">Bookshelf ID: NBK604939</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/39013055" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">39013055</a></span>DOI: <a href="http://dx.crossref.org/10.17917/YTHD-KF51" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/YTHD-KF51</a></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/nciprotocols/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/nciprotocols/ncipr42/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/nciprotocols/ncipr38/" title="Next page in this title">Next ></a></div></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK604939/?report=reader">PubReader</a></li><li><a href="/books/NBK604939/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK604939" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK604939" style="display:none" title="Cite this Page"><div class="bk_tt">Neun BW, Cedrone E, Dobrovolskaia MA. Mouse Granulocyte-Macrophage Colony-Forming Unit Assay: Version 4. 2020 May. In: National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. NCL Method ITA-3.<span class="bk_cite_avail"></span> doi: 10.17917/YTHD-KF51</div></div></li><li><a href="/books/NBK604939/pdf/Bookshelf_NBK604939.pdf">PDF version of this page</a> (910K)</li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ncipr39.s1" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ncipr39.s2" ref="log$=inpage&link_id=inpage">Principles</a></li><li><a href="#ncipr39.s3" ref="log$=inpage&link_id=inpage">Reagents, Materials, and Equipment</a></li><li><a href="#ncipr39.s4" ref="log$=inpage&link_id=inpage">Animals</a></li><li><a href="#ncipr39.s5" ref="log$=inpage&link_id=inpage">Reagent and Control Preparation</a></li><li><a href="#ncipr39.s6" ref="log$=inpage&link_id=inpage">Preparation of Study Samples</a></li><li><a href="#ncipr39.s7" ref="log$=inpage&link_id=inpage">Isolation and Counting of Bone Marrow Cells</a></li><li><a href="#ncipr39.s8" ref="log$=inpage&link_id=inpage">Experimental Procedure</a></li><li><a href="#ncipr39.s9" ref="log$=inpage&link_id=inpage">Description of CFU-GM</a></li><li><a href="#ncipr39.s10" ref="log$=inpage&link_id=inpage">Calculations</a></li><li><a href="#ncipr39.s11" ref="log$=inpage&link_id=inpage">Acceptance Criteria</a></li><li><a href="#ncipr39.rl.r1" ref="log$=inpage&link_id=inpage">References</a></li><li><a href="#ncipr39.s12" ref="log$=inpage&link_id=inpage">Abbreviations</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=5641150" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=5641150" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/17318617" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Effect of the Cdk-inhibitor roscovitine on mouse hematopoietic progenitors in vivo and in vitro.</a><span class="source">[Cancer Chemother Pharmacol. 2007]</span><div class="brieflinkpop offscreen_noflow">Effect of the Cdk-inhibitor roscovitine on mouse hematopoietic progenitors in vivo and in vitro.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Song H, Vita M, Sallam H, Tehranchi R, Nilsson C, Sidén A, Hassan Z. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Cancer Chemother Pharmacol. 2007 Nov; 60(6):841-9. Epub 2007 Feb 22.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/7561684" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha.</a><span class="source">[J Exp Med. 1995]</span><div class="brieflinkpop offscreen_noflow">Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Young JW, Szabolcs P, Moore MA. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Exp Med. 1995 Oct 1; 182(4):1111-9. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/8542927" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">In vitro sensitivity of post-bone marrow transplantation CFU-GM and BFU-E to TNF-alpha and IFN-gamma.</a><span class="source">[Exp Hematol. 1995]</span><div class="brieflinkpop offscreen_noflow">In vitro sensitivity of post-bone marrow transplantation CFU-GM and BFU-E to TNF-alpha and IFN-gamma.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Papadakis V, Ferguson KF, Heller G, Kernan NA. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Exp Hematol. 1995 Dec; 23(14):1422-30. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/8542941" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Proliferative response of human marrow myeloid progenitor cells to in vivo treatment with granulocyte colony-stimulating factor alone and in combination with interleukin-3 after autologous bone marrow transplantation.</a><span class="source">[Exp Hematol. 1995]</span><div class="brieflinkpop offscreen_noflow">Proliferative response of human marrow myeloid progenitor cells to in vivo treatment with granulocyte colony-stimulating factor alone and in combination with interleukin-3 after autologous bone marrow transplantation.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Lemoli RM, Fortuna A, Fogli M, Gherlinzoni F, Rosti G, Catani L, Gozzetti A, Miggiano MC, Tura S. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Exp Hematol. 1995 Dec; 23(14):1520-6. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/39013056" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Radioactive Blood Partitioning Assay: Version 1.1.</a><span class="source">[National Cancer Institute’s Na...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Radioactive Blood Partitioning Assay: Version 1.1.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Stern ST. </em><em 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