{"Equipment":"<strong>Equipment</strong><ul>    <li>Scissors and forceps for biopsy</li>    <li>Precision balance</li>    <li>Calibrated single and multichannel pipettes</li>    <li>Plate shaker</li>    <li>Refrigerated centrifuge</li>    <li>Flow Cytometer (capable of distinguishing a minimum of 8 colours per well)</li>    <li>Tissue dissociator:</li>    <ul>        <li>GentleMACS tissue dissociator <strong><em>OR</em></strong></li>        <li>Equipment for manual dissociation</li>    </ul>    <li>Cell counter equipment:</li>    <ul>        <li>Orflo Moxi-Z Cell counter <strong><em>OR</em></strong></li>        <li>Coulter Vicell XR OR Life Technologies Attune&reg; Flow Cytometer <strong><em><span style=\"color: black;\">OR</span></em></strong></li>        <li>Haemocytometer</li>    </ul></ul><strong>Supplies</strong><ul>    <li>96-well V-bottomed plates (Falcon <span style=\"font-family: Symbol;\">#</span>353263)</li>    <li>Petri dishes</li>    <li>Dispensing troughs</li>    <li>Low retention pipette tips for antibody solutions</li>    <li><em>(if using GentleMACS for dissociation)</em> C Tubes. It is acceptable to re-use these once.</li>    <li>50ml Falcon tubes</li>    <li>Cell strainers e.g. 70&mu;m cell strainers that fit 50ml Falcon tubes (BD Falcon, <span style=\"font-family: Symbol;\">#</span>352350) <strong><em>OR</em></strong> 70-80&micro;M Nytex</li>    <li>Cell counter recipients (i.e., slides/cassettes/etc. for cell counter)</li>    <li><em>(if sample processing delayed)</em> RPMI 1640</li>    <li><em>(if sample processing on same day)</em> HBSS, with phenol red</li>    <li>CS (calf serum)</li>    <li>PBS with Mg<sup>2+</sup>, with Ca<sup>2+</sup> (for <u>enzyme buffer</u> used for DNAse and Collagenase D digestions)</li>    <li>PBS without Mg<sup>2+</sup>, without Ca<sup>2+</sup> (for <u>FACS buffer</u> to be used in all steps subsequent to enzymatic digest)</li>    <li>EDTA (0.5M stock; final concentration 2mM)</li>    <li>Digestion enzyme (Collagenase D from Roche <span style=\"font-family: Symbol;\">#</span>11088858001), stock solution in <u>enzyme buffer</u> (see below), aliquoted and stored at &ndash;20&deg;C</li>    <li>DNAse I stock solution (Sigma, <span style=\"font-family: Symbol;\">#</span>DN25) in <u>enzyme buffer</u> (see below), aliquoted and stored at -20&deg;C</li>    <li>RBC lysis buffer (eBioscience <span style=\"font-family: Symbol;\">#</span>00-4300-54 or BD Biosciences <span style=\"font-family: Symbol;\">#</span>555899, both 10X from manufacturer)</li>    <li>HEPES (pH 7.2-7.4)</li></ul>","Procedure":"<p>This protocol requires several steps in the collection, preparation and analysis of the samples. Each one is detailed separately below.</p><p><strong>Reagent preparation</strong></p><p><em>Note that two different PBS solutions are required for the protocol below, one with Ca<sup>2+</sup> and Mg<sup>2+</sup>, another without Ca<sup>2+</sup> and Mg<sup>2+</sup>.</em></p><ul>    <li><strong>Collection buffer:</strong></li>    <ul>        <li>(<em>if spleens are to be processed on the same day</em>) HBSS with Ca<sup>2+</sup> and Mg<sup>2+</sup> and phenol red (e.g. Life Technologies 14170161) <strong><em>OR</em></strong></li>        <li>(<em>if analysis will be delayed</em>) RPMI medium with 2% CS added.</li>    </ul>    <li><strong>FACS buffer</strong> (<span style=\"color: black;\">for all steps subsequent to enzymatic digest</span>; stable for up to 1 month in the fridge):</li>    <ul>        <li>PBS 1X <em><u>without</u></em> Ca<sup>2+</sup>/Mg<sup>2+</sup> <strong><em>OR</em></strong></li>        <li>HBSS 1X <em><u>without</u></em> Ca<sup>2+</sup>/Mg<sup>2+</sup></li>        <li>EDTA 2mM</li>        <li>2% (v/v) CS</li>        <li>10mM HEPES, pH 7.2-7.4</li>    </ul>    <li><strong>Brilliant Stain Buffer</strong> (BD 563794; for all steps when two or more brilliant violet antibodies are used to prevent non-specific dye-to-dye interaction)</li>    <li><strong>Enzyme buffer </strong>(<span style=\"color: black;\">for DNAse and Collagenase D digestions; </span>Stable for up to 1 month in the fridge<span style=\"color: black;\">)</span>:</li>    <ul>        <li>PBS <em><u>with</u></em> Ca<sup>2+</sup> and Mg<sup>2+</sup> <strong><em>OR</em></strong></li>        <li>HBSS 1X <em><u>with</u></em> Ca<sup>2+</sup>/Mg<sup>2+</sup></li>        <li>2% (v/v) CS;</li>        <li>10mM HEPES, pH 7.2-7.4</li>    </ul>    <li><strong>RBC Lysis buffer</strong>: Prepare a 1X solution in ddH<sub>2</sub>0 from 10X stock lysis buffer.</li>    <li><strong>Stopping buffer </strong>(require 300 <span style=\"color: black;\">&micro;</span>l per sample):</li>    <ul>        <li>1x PBS without Ca<sup>2+</sup> and Mg<sup>2+</sup> or 1X HBSS without Ca<sup>2+</sup> and Mg<sup>2+</sup></li>        <li>0.1 M EDTA (37.5 g/L)</li>    </ul></ul><ul><li><strong>Antibody cocktails for Panels 1 &amp; 2</strong><ul><li>Protect antibodies and prepared cocktails from direct light.</li><li>Final concentration of antibodies should be determined by titration to ensure saturating amounts of antibody are used. Appropriate amounts of antibodies can be mixed together from the manufacturer&rsquo;s stock solutions and stored for 1 week at 4&ordm;C prior to dilution in FACS buffer immediately before use. Do NOT pre-mix BV antibodies. These should be added fresh to the diluted staining mixure.</li><li>Each sample will require 50 &micro;l (or up to 100 &micro;l) of diluted 1X antibody cocktail.</li><li>Antibody cocktails should be gently but thoroughly mixed to ensure homogeneity of the solutions.</li><li>In order to eliminate aggregated antibodies from your mix, centrifuge each antibody cocktail for 8 min at 20,000x<em>g</em> and 8&deg;C prior to staining cells.</li></ul></li></ul><ul><li><strong>Antibody Panels</strong></li></ul><ul><li>Recommended antibody (marker) panels, Panel A for T, NKT and NK cells, Panel B for B, myeloid and NK cells are shown below, along with optional markers that may be used by some centres. Core antibodies are required for upload of data; optional markers are not and are listed in alphabetical order. Clones and fluorochromes used should be uploaded for required and optional markers. Where not indicated, clone and fluorochrome choice is dependent on available detectors and filters on the cytometer used at each centre.</li></ul><p><em>Panel A</em></p><table style=\"margin-left: 36.0pt; border-collapse: collapse; border: none;\"><thead><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-right: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p><strong><span style=\"color: white;\">Type</span></strong></p></td><td style=\"width: 104.7pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p><strong><span style=\"color: white;\">Antibody (Marker)</span></strong></p></td><td style=\"width: 100.55pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p><strong><span style=\"color: white;\">Clone</span></strong></p></td><td style=\"width: 106.65pt; border: solid black 1.0pt; border-left: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p><strong><span style=\"color: white;\">Fluorochrome</span></strong></p></td></tr></thead><tbody><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD5</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>53-7.3</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>BV421</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD4</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>RM4-5</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>FITC</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD44</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>IM7</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>PE</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD8a</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>53-6.7</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>PE-CF594</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD25</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>PC61</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>PE-Cy7</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD161</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>PK136</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>APC</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD62L</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>MEL-14</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>APC-Cy7</p></td></tr><tr><td style=\"width: 102.9pt; border-top: none; border-left: solid black 1.0pt; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>Live/Dead</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>-</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>SytoxBlue</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD3e</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>145-2C11</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD24</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>M1/69</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD27</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>LG.3A10</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD357/GITR</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>DTA-1</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD45</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>30-F11</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>KLRG1</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>2F1</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>Ly6c</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>AL-21</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>TCRd</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>GL-3</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr></tbody></table><p>&nbsp;</p><p><em>Panel B</em></p><table style=\"margin-left: 36.0pt; border-collapse: collapse; border: none;\"><thead><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-right: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p><strong><span style=\"color: white;\">Type</span></strong></p></td><td style=\"width: 104.7pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p><strong><span style=\"color: white;\">Antibody (Marker)</span></strong></p></td><td style=\"width: 100.55pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p><strong><span style=\"color: white;\">Clone</span></strong></p></td><td style=\"width: 106.65pt; border: solid black 1.0pt; border-left: none; background: #365F91; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p><strong><span style=\"color: white;\">Fluorochrome</span></strong></p></td></tr></thead><tbody><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD5</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>53-7.3</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>BV421</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>Ly6G</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>1A3</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>BV421</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD19</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>1D3</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>BV510</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>Ly6C</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>AL-21</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>FITC</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD21/CD35</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>7G6</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>PE</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD11b</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>M1/70</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>PE-CF594</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD11c</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>HL3</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>PE-Cy7</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD161</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>PK136</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>APC</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>MHCII</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>M5/114.15.2</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>APC-Cy7 or A700</p></td></tr><tr><td style=\"width: 102.9pt; border-top: none; border-left: solid black 1.0pt; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Required</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>Live/Dead</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>-</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 3.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>SytoxBlue</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD23</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>B3B4</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD27</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>LG3.A10</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD43</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>S7</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD44</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>&nbsp;</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD45</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>30-F11</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>CD317</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>927</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>F4/80</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>BM8</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>IgD</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>&nbsp;</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr><tr><td style=\"width: 102.9pt; border: solid black 1.0pt; border-top: none; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"137\"><p>Optional</p></td><td style=\"width: 104.7pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"140\"><p>KLRG</p></td><td style=\"width: 100.55pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"134\"><p>2F1</p></td><td style=\"width: 106.65pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: 0cm 5.4pt 0cm 5.4pt;\" width=\"142\"><p>&nbsp;</p></td></tr></tbody></table><p>&nbsp;</p><ul><li><strong>Read buffer / dead cell exclusion dye</strong></li></ul><ul><ul><li>SytoxBlue at 1:10000 concentration in <u>FACS buffer</u> <strong><em>OR</em></strong></li><li>SytoxGreen at 1:20000 concentration in <u>FACS buffer</u></li><li>&nbsp;Zombie Near Infra-Red live dead from Biolegend at 1:2000 concentration</li><li>Require 200 &mu;l per well (i.e. 400 &mu;l for each spleen).</li></ul><li><strong>Enzyme cocktail (working solution): </strong>3 ml for each spleen, containing final concentrations of:</li><ul><li>DNAse I: 30-100 &mu;g (from 10 mg/ml stock in enzyme buffer stored in single experiment aliquots at -20&ordm;C, do not freeze-thaw stock)</li><li>Collagenase D: 600 Mandl Units (from 30 U/&micro;l stock in enzyme buffer stored in single experiment aliquots at -20&ordm;C, do not freeze-thaw stock)</li></ul></ul><p><strong><em>NOTE</em></strong><strong>: </strong>To top up to the 3ml use enzyme buffer; any intermediate dilutions of the enzyme stock solutions should be prepared with <u>enzyme buffer</u>.</p><p><strong>Other preparations on the day</strong></p><ul><li>Bring RBC lysis buffer and stop solution to room temperature<span style=\"color: #76923c;\">.</span></li><li>Prepare wet ice box, label tubes, etc.</li></ul><p><strong><u>Note</u></strong><strong> all centrifuge steps are: 5 min, 400 x g at 8&deg;C</strong></p><p><strong>Spleen collection</strong></p><ul><li>Collect the spleen from euthanized mice.</li><li>Remove all fat from the spleen and weigh the organ on a petri dish (do not hydrate the organ before weighing it as this would lead to substantial errors in measurement).</li><li>Place the spleen in a 1.5ml eppendorf tube with 1 mL of sample collection buffer on ice. Use:</li><ul><li><em>(if spleens are to be processed on the same day)</em> HBSS without calcium, without magnesium but with phenol red <strong><em>OR</em></strong></li><li><em>(if analysis will be delayed)</em> RPMI with 2% CS buffer.</li></ul></ul><p><strong>Spleen dissociation / digests</strong></p><p><strong>If using a GentleMacs tissue dissociator:</strong></p><ul><li>Add the spleen to a GentleMACS C tube containing 3 ml of 1X enzyme cocktail.</li><li>Clip the tube on GentleMACS dissociator and run programme spleen_2.</li><li>Incubate cell suspension for 30 minutes with gentle mixing at least every 5 minutes. Register incubation temperature.</li><li>Run programme spleen_3.</li><li>Add 300 &mu;L of stopping buffer and mix by inversion to block enzymatic digestion and dissociate T cell-dendritic cell interactions.</li><li>Filter cell suspension:</li><ul><li>&nbsp;through 70-80 &mu;m Nylon mesh filter into a 50 mL Falcon tube <strong><em>OR</em></strong></li><li>directly from C-tubes pour splenocyte suspension through 30 <span style=\"font-family: Symbol;\">m</span>m CellTrics Partec filters (<span style=\"font-family: Symbol;\">#</span>04-0042-2316) into 15 ml tubes.</li></ul><li><em>(optional)</em> Wash the GentleMACS C tube with 5ml <u>FACS buffer</u>, filter and pool with flow-through from previous step.</li><li>Centrifuge for 5 minutes, 400 x g at 8&deg;C and discard supernatant.</li><li>Resuspend total splenocytes in 1 mL cold <u>FACS buffer</u> and keep on ice (this step is not required if counting is performed on the attune).</li></ul><p><strong>OR, if performing manual digests:</strong></p><ul><li>Place weighed spleen in 12x75mm tube containing 1ml of collagenase solution in 1X HBSS with Ca<sup>2+</sup> and Mg<sup>2+</sup> (17-0.2 W&uuml;nsch unit/ml)</li><li>Mince into fine pieces using small scissors, place on ice until all samples are minced.</li><li>Add 2ml collagenase (17-0.2 W&uuml;nsch unit/ml) to each tube and place in a 37&ordm;C water bath for 30 minutes.</li><li>Tricturate (pipetting vigorously up and down using a 1 mL pipetman) the mixture to break up clumps.</li><li>Spin at 500 x g in a swing bucket rotor for 5 min at 10&ordm;C. Decant the supernatant, rack the tubes or vortex to resuspend the pellet. Add 2ml <u>FACS buffer</u>, mix well by vortexing, take 10 &micro;l for the counting step.</li><li>Dilutions for counting: 2 serial 1:10 dilutions (10&micro;l cells + 90&micro;l <u>FACS buffer</u>, then 10&micro;l of the 1:10 dilution + 90&micro;l buffer.)</li><li>Spin for 5min, 500 x g at 10&ordm;C, decant supernatant, blot the top of the tube, resuspend pellet at 1x10<sup>8</sup> cells/ml.</li></ul><p><strong>Cell counting</strong></p><ul><li>Perform a cell count on an aliquot of the re-suspended cells (adjust concentration according to the cell counter method used).</li><li>Note the cell count, correct for dilution and calculate the concentration in cells per &micro;l.</li><li>Cell count:</li><ul><li><em><u>If performed before RBC lysis</u></em>, pipette the volume containing approximately 4 million cells/well to a 96 well plate in horizontal fashion starting from A1 onwards for panel 1 staining.</li><li><em><u>If performed after RBC lysis</u></em>, pipette the volume containing approximately 1-2 million cells/well to a 96 well plate in horizontal fashion starting from A1 onwards for panel 1 staining.</li></ul><li>Do the same for panel 2 staining in separate wells leaving a few empty rows between the panels to avoid cross contamination.</li><li>Top up to final volume of 100 <span style=\"font-family: Symbol;\">m</span>l using <u>FACS buffer</u>, centrifuge, discard supernatant and keep plate on wet ice.</li></ul><p><strong>Red blood cell lysis, blocking &amp; staining</strong></p><ul><li>Remove plate from ice and add 30 to 100 <span style=\"font-family: Symbol;\">m</span>l of 1X RBC lysis buffer (at room temperature) to each cell pellet from the previous step.</li><li>Pipette up and down 2-3 times to break up the pellet and ensure complete lysis. Alternatively, vortex the edges of the plates, then pipet quickly once to ensure resuspension is ideal for optimal lysis.</li><li>Incubate for 1 minute at room temperature and then return to ice and add 100 to 200 <span style=\"font-family: Symbol;\">m</span>l of <u>FACS buffer</u> (to stop lysis) to each well.</li></ul><p><strong>Note</strong>: <em>Following RBC lysis, every centrifugation step can be performed at 2000rpm for 1 minute in a 96 well plate, which significantly speeds up the protocol.</em> <em>Do take care to resuspend the cells very well to prevent HTS clumping.</em></p><ul><li>Centrifuge, discard supernatant and resuspend in 200 <span style=\"font-family: Symbol;\">m</span>l <u>FACS buffer</u> (this step is not required if lysis was performed in 30 &micro;l, since there will be enough volume left in the well for a bigger wash of 200 &micro;l; saves time on a spin).</li><li>Again centrifuge and discard supernatant and resuspend in 50 <span style=\"font-family: Symbol;\">m</span>l of 1:100 Fc block and incubate on ice for 10 min. Top up to 200 <span style=\"font-family: Symbol;\">m</span>l using <u>FACS buffer</u> after incubation.</li><li>Take antibody (AB) cocktails from the fridge.&nbsp; In order to eliminate aggregated ABs from your mix before use, centrifuge each AB cocktail for 8 min at 20,000 x g and 4&deg;C. Dilute antibody cocktail to final working concentration with FACS buffer, or Brilliant stain buffer when two or more brilliant violet antibodies are used, to make the AB mix.</li><li>Centrifuge plate, discard supernatant and resuspend in 50 to 100 <span style=\"font-family: Symbol;\">m</span>l 1X AB mix in appropriate wells for individual panels followed by incubation on ice and in the dark for 20 min.</li><li><u>If using Sytox Blue/Sytox Green as live/dead discriminator:</u></li><ul><li>Top up to 200 <span style=\"font-family: Symbol;\">m</span>l with <u>FACS or Brilliant Stain buffer</u> after incubation. Centrifuge, discard supernatant and resuspend in 200 <span style=\"font-family: Symbol;\">m</span>l <u>FACS or Brilliant Stain buffer</u>.</li><li>When ready to read plate, centrifuge again and discard supernatant. Resuspend the pellet in 200 <span style=\"font-family: Symbol;\">m</span>l of read buffer (Sytox Blue diluted 1:10000 in <u>FACS buffer</u>; Sytox Green diluted 1:20000 in<u> FACS buffer</u>).</li></ul></ul><ul><li><u>If using Zombie NIR dye as live/dead discriminator</u>:<ul><li>Add 200 <span style=\"font-family: Symbol;\">m</span>l of PBS (RT) to all samples</li><li>Spin at 2000 rpm for 1 minute 8<span style=\"font-family: Symbol;\">&deg;</span>C</li><li>Add 100 <span style=\"font-family: Symbol;\">m</span>l/well of Zombie Near-IR Live/Dead dye (1/2000) made up in PBS incubate at room temperature for 10 mins, add 200 <span style=\"font-family: Symbol;\">m</span>l <u>FACS buffer</u>.</li></ul></li></ul><p><strong>General Recommendations for Setting up Cytometer</strong></p><p>Set up the analyser to aim acquire 300,000 viable events (live cells) for each of Panels 1 and 2. 500,000 are recommended for panel 2 in order to increase robustness of myeloid population assessment for low frequency populations (macrophages, DCs).</p>","Purpose":"<p>This test differentiates immune cell sub-populations via flow cytometry.<p></p>Description: increased CD4-positive T cell number (MP:0008074), decreased CD4-positive T cell number (MP:0008075), etc., ...</p>","Experimental Design":"<ul>     <li><strong>Minimum number:</strong> 3M + 3F</li>     <li><strong>Age at test:</strong>  Week 59</li>     <li><strong>Sex:</strong> Both (sexually dimorphic)</li> </ul>","Notes":"<p><strong>Visual help for Gating</strong></p><img src=\"https://api.mousephenotype.org/impress/image/9bbafbea617a8d19e374366a7943ad576c00d693f492a0a39af7b31f6706cc3a\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/ba80ea5ed2029b73c622bee27c8f5166cf0d1b8ce804392a2c34a85a21d93a3a\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/ad01d7c421a120ba1e18b832497268a78dd61cbab3341894fd1d037d58ac8db4\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/a8e1d1a2976264f27d760ee3f29a8f8a4c60205bc2bb3fdf1da810c9d564266e\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/a6163c117761fb7145401d552c4f35bef2ee04908aba90ac851177dc84b86e59\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/2efdf3fe7ca32cedd42c0c9c05c5e6423035419013b037faf4d6280722dce41e\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/da44964ea7d159bb82ff14209dc779f5fd61aac2c4de123a4b65ab4027e55dc4\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/907d66f982c0f9ba08fbaacb1b99286dae3585b6afaa5a3749119660e8788c15\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/6b0f4cadbac152ead1bf6123b2677b03ce85b07c19b1772d04f9a1e4f5e4be46\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/5082e9f5ad24c58583c65da1bafa355441349b399704063377549bfebcbe3a38\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/6342bb145ed2abcbbce80e0cb03d83a35842a5d53dd1d1957f213245bdba6d2a\"> <br> <img src=\"https://api.mousephenotype.org/impress/image/cc188cd60c99db9f7da1fc23a4fb2d8c3b072d94aad7ced634afaa52c141f307\">"}