{"Equipment":"<p>\n\t<strong>Equipment</strong></p>\n<ul>\n\t<li>\n\t\tScissors and forceps for biopsy</li>\n\t<li>\n\t\tPrecision balance</li>\n\t<li>\n\t\tCalibrated single and multichannel pipettes</li>\n\t<li>\n\t\tPlate shaker</li>\n\t<li>\n\t\tRefrigerated centrifuge</li>\n\t<li>\n\t\tFlow Cytometer (capable of distinguishing a minimum of 8 colours per well)</li>\n\t<li>\n\t\tTissue dissociator:\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tGentleMACS tissue dissociator <strong><em>OR</em></strong></li>\n\t\t\t<li>\n\t\t\t\tEquipment for manual dissociation</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\tCell counter equipment:\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tOrflo Moxi-Z Cell counter <strong><em>OR</em></strong></li>\n\t\t\t<li>\n\t\t\t\tCoulter Vicell XR OR Life Technologies Attune&reg; Flow Cytometer</li>\n\t\t</ul>\n\t</li>\n</ul>\n<p>\n\t<strong>Supplies</strong></p>\n<ul>\n\t<li>\n\t\t96-well V-bottomed plates (Falcon #353263)</li>\n\t<li>\n\t\tPetri dishes</li>\n\t<li>\n\t\tDispensing troughs</li>\n\t<li>\n\t\tExtra long 10 &micro;l pipette tips for antibody solutions</li>\n\t<li>\n\t\t<em>(if using GentleMACS for dissociation)</em> C Tubes. It is acceptable to re-use these once.</li>\n\t<li>\n\t\t50ml Falcon tubes</li>\n\t<li>\n\t\tCell strainers e.g. 70&mu;m cell strainers that fit 50ml Falcon tubes (BD Falcon, #352350) <strong><em>OR</em></strong> Nytex</li>\n\t<li>\n\t\tCell counter <a>recipients (i.e., slides/cassettes/etc. for cell counter)</a></li>\n\t<li>\n\t\t<em>(if sample processing delayed)</em> RPMI 1640</li>\n\t<li>\n\t\t<em>(if sample processing on same day)</em> <a>HBSS&nbsp;</a>(with phenol red)</li>\n\t<li>\n\t\t<a>CS (calf serum)</a></li>\n\t<li>\n\t\tPBS with Mg2+, with Ca2+ (for <u>enzyme&nbsp;buffer</u> used for DNAse and Collagenase D digestions)</li>\n\t<li>\n\t\tPBS without Mg2+, without Ca2+ (for <u>FACS buffer</u> to be used in all steps subsequent to enzymatic digest)</li>\n\t<li>\n\t\tEDTA (final concentration 2mM)</li>\n\t<li>\n\t\tDigestion enzyme (Collagenase D from&nbsp;Roche, #11088858001) stock solution in <u>enzyme&nbsp;buffer</u>&nbsp;(see below), aliquoted and stored at &ndash;20&deg;C</li>\n\t<li>\n\t\tDNAse I stock solution (Sigma, #DN25) in <u>enzyme&nbsp;buffer</u>&nbsp;(see below), aliquoted and stored at -20&deg;C</li>\n\t<li>\n\t\tRBC lysis buffer (eBioscience #00-4300-54 or BD Biosciences #555899, both 10X from manufacturer)</li>\n\t<li>\n\t\t<a>HEPES</a> (pH 7.2)</li>\n</ul>\n","Procedure":"<p>\n\tThis protocol requires several steps in the collection, preparation and analysis of the samples. Each one is detailed separately below.</p>\n<p>\n\t<strong>Reagent preparation</strong></p>\n<p>\n\t<em>Note that two different PBS solutions are required for the protocol below, one with Ca2+ and with Mg2+, another without Ca2+ and without Mg2+.</em></p>\n<ul>\n\t<li>\n\t\t<strong>Collection buffer:</strong>\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\t(<em>if spleens are to be processed on the same day</em>) HBSS with Ca2+/Mg2+ and phenol red (Life Technologies 14170161; check if it has phenol red) <strong><em>OR</em></strong></li>\n\t\t\t<li>\n\t\t\t\t(<em>if analysis will be delayed</em>) RPMI medium with 2% CS added.</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\t<strong>FACS buffer</strong> (for all steps subsequent to enzymatic digest; stable for up to 1 month in the fridge):\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tPBS 1X <em><u>without</u></em> Ca2+/Mg2+ <strong><em>OR</em></strong></li>\n\t\t\t<li>\n\t\t\t\tHBSS 1X <em><u>without</u></em> Ca2+/Mg2+</li>\n\t\t\t<li>\n\t\t\t\tEDTA 2mM</li>\n\t\t\t<li>\n\t\t\t\t2% CS (v/v)</li>\n\t\t\t<li>\n\t\t\t\t10mM HEPES</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\t<strong>Enzyme buffer </strong>(for DNAse and Collagenase D digestions; Stable for up to 1 month in the fridge):\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tPBS <em><u>with</u></em> Ca2+ and Mg2+ <strong><em>OR</em></strong></li>\n\t\t\t<li>\n\t\t\t\tHBSS 1X <em><u>with</u></em> Ca2+/Mg2+</li>\n\t\t\t<li>\n\t\t\t\t2% CS (v/v);</li>\n\t\t\t<li>\n\t\t\t\t10mM HEPES</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\t<strong>RBC Lysis buffer</strong>: Prepare a 1X solution in ddH<sub>2</sub>0 from lysis buffer.</li>\n\t<li>\n\t\t<strong>Stopping buffer&nbsp;</strong>(require 300 &micro;l per sample):\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\t1x PBS without Ca2+ and without Mg2+ or HBSS</li>\n\t\t\t<li>\n\t\t\t\t0.1 M EDTA (37.5 g/L)</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\t<strong>Antibody cocktails for Panels 1 &amp; 2</strong>\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tProtect antibodies and prepared cocktails from direct light.</li>\n\t\t\t<li>\n\t\t\t\tMastermix concentration, storage temperature and stability to be determined after panels 1 and 2 have been finalised and tested.</li>\n\t\t\t<li>\n\t\t\t\tEach sample will require 50 &micro;l (or up to 100 &micro;l) of diluted 1X antibody cocktail.</li>\n\t\t\t<li>\n\t\t\t\tAntibody cocktails should be gently but thoroughly mixed or quickly vortexed to ensure homogeneity of the solutions.</li>\n\t\t\t<li>\n\t\t\t\tIn order to eliminate aggregated antibodies from your mix, centrifuge each antibody cocktail for 8 min at 20,000xg and 8&deg;C prior to staining cells.</li>\n\t\t</ul>\n\t</li>\n</ul>\n<ul>\n\t<li>\n\t\t<strong>Read buffer / dead cell exclusion dye</strong>\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tSytoxBlue at 1:10000 concentration in <u>FACS buffer</u> <strong><em>OR</em></strong></li>\n\t\t\t<li>\n\t\t\t\tSytoxGreen at 1:20000 concentration in <u>FACS buffer</u></li>\n\t\t\t<li>\n\t\t\t\t&nbsp;Zombie Near Infra-Red live dead from Biolegend at 1:2000 concentration</li>\n\t\t\t<li>\n\t\t\t\tRequire 200 &mu;l per well (i.e. 400 &mu;l for each spleen).</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\t<strong>Enzyme cocktail (working solution): </strong>3 ml per each spleen, containing final concentrations of:\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tDNAse I: 30 &mu;g</li>\n\t\t\t<li>\n\t\t\t\tCollagenase D: 600 Mandl Units</li>\n\t\t</ul>\n\t</li>\n</ul>\n<p style=\"margin-left: 80px;\">\n\t<strong><em>NOTE</em></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>\n<p>\n\t<strong>Other preparations on the day</strong></p>\n<ul>\n\t<li>\n\t\tBring RBC lysis buffer and stop solution to room temperature.</li>\n\t<li>\n\t\tPrepare wet ice box, label tubes, etc.</li>\n</ul>\n<p>\n\t<strong><u>Note</u></strong><strong> all centrifuge steps are: 5 min, 400 x g at 8&deg;C</strong></p>\n<p>\n\t<strong>Spleen collection</strong></p>\n<ul>\n\t<li>\n\t\tCollect the spleen from euthanized mice.</li>\n\t<li>\n\t\tRemove 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>\n\t<li>\n\t\tPlace the spleen in a 1.5ml eppendorf tube with 1 mL of sample collection buffer on ice. Use:\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\t<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>\n\t\t\t<li>\n\t\t\t\t<em>(if analysis will be delayed)</em> RPMI with 2% CS buffer.</li>\n\t\t</ul>\n\t</li>\n</ul>\n<p>\n\t<strong>Spleen dissociation / digests</strong></p>\n<p style=\"margin-left:36pt;\">\n\t<strong>If using a GentleMacs tissue dissociator:</strong></p>\n<ul>\n\t<li>\n\t\tAdd the spleen to a GentleMACS C tube containing 3 ml of 1X enzyme cocktail.</li>\n\t<li>\n\t\tClip the tube on GentleMACS dissociator and run programme spleen_2.</li>\n\t<li>\n\t\tIncubate cell suspension for 30 minutes with gentle mixing at least every 5 minutes. Register incubation temperature.</li>\n\t<li>\n\t\tRun programme spleen 3.</li>\n\t<li>\n\t\tAdd 300 &mu;L of stopping buffer and mix by inversion to block enzymatic digestion and dissociate T cell-dendritic cell interactions.</li>\n\t<li>\n\t\tFilter cell suspension:\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\t&nbsp;through 70 &mu;m Nylon mesh filter into a 50 mL Falcon tube <strong><em>OR</em></strong></li>\n\t\t\t<li>\n\t\t\t\tdirectly from C-tubes pour splenocyte suspension through 30 mm CellTrics Partec filters (#04-0042-2316) into 15 ml tubes.</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\t<em>(optional)</em> Wash the GentleMACS C tube with 5ml <u>FACS buffer</u>, filter and pool with flow-through from previous step.</li>\n\t<li>\n\t\tCentrifuge for 5 minutes, 400 x g at 8&deg;C and discard supernatant.</li>\n\t<li>\n\t\tResuspend 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>\n</ul>\n<p style=\"margin-left:36pt;\">\n\t<strong>OR, if performing manual digests:</strong></p>\n<ul>\n\t<li>\n\t\tPlace weighed spleen in 12x75mm tube containing 1ml of collagenase&nbsp; solution in 1X HBSS with Ca2+ and Mg2+ (0.17-0.2 W&uuml;nsch unit/ml)</li>\n\t<li>\n\t\tMince into fine pieces using small scissors, place on ice until all samples are minced.</li>\n\t<li>\n\t\tAdd 2ml collagenase (0.17-0.2 W&uuml;nsch unit/ml) to each tube and place in a 37&ordm;C water bath for 30 minutes.</li>\n\t<li>\n\t\tTricturate (pipetting vigorously up and down using a 1 mL pipetman) the mixture to break up clumps.</li>\n\t<li>\n\t\tSpin 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>\n\t<li>\n\t\tDilutions 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>\n\t<li>\n\t\tSpin 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>\n</ul>\n<p>\n\t<strong>Cell counting</strong></p>\n<ul>\n\t<li>\n\t\tPerform a cell count on an aliquot of the re-suspended cells (adjust concentration according to the cell counter method used).</li>\n\t<li>\n\t\tNote down the cell count, correct for dilution and calculate the concentration in cells per &micro;l.</li>\n\t<li>\n\t\tCell count:\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\t<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>\n\t\t\t<li>\n\t\t\t\t<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>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\tDo the same for panel 2 staining in separate wells leaving a few empty rows between the panels to avoid cross contamination.</li>\n\t<li>\n\t\tTop up to final volume of 100 ml using <u>FACS buffer</u>, centrifuge, discard supernatant and keep plate on wet ice.</li>\n</ul>\n<p>\n\t<strong>Red blood cell lysis, blocking &amp; staining</strong></p>\n<ul>\n\t<li>\n\t\tRemove plate from ice and add 30 to 100 ml of 1X RBC lysis buffer (at room temperature) to each cell pellet from the previous step.</li>\n\t<li>\n\t\tPipette 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>\n\t<li>\n\t\tIncubate for 1 minute at room temperature and then return to ice and add 100 to 200 ml of <u>FACS buffer</u> (to stop lysis) to each well.</li>\n</ul>\n<p style=\"margin-left:18pt;\">\n\t<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>\n<ul>\n\t<li>\n\t\tCentrifuge, discard supernatant and resuspend in 200 ml <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>\n\t<li>\n\t\tAgain centrifuge and discard supernatant and resuspend in 50 ml of 1:100 Fc block and incubate on ice for 10 min. Top up to 200 ml using <u>FACS buffer</u> after incubation.</li>\n\t<li>\n\t\tTake 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.</li>\n\t<li>\n\t\tCentrifuge plate, discard supernatant and resuspend in 50 to 100 ml 1X AB mix in appropriate wells for individual panels followed by incubation on ice and in the dark for 20 min.</li>\n\t<li>\n\t\t<u>If using Sytox Blue/Sytox Green as live/dead discriminator:</u>\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tTop up to 200 ml with <u>FACS buffer</u> after incubation. Centrifuge, discard supernatant and resuspend in 200 ml <u>FACS buffer</u>.</li>\n\t\t\t<li>\n\t\t\t\tWhen ready to read plate, centrifuge again and discard supernatant. Resuspend the pellet in 200 ml of read buffer (Sytox Blue diluted 1:10000 in <u>FACS buffer</u>; Sytox Green diluted 1:20000 in <u>FACS buffer</u>).</li>\n\t\t</ul>\n\t</li>\n\t<li>\n\t\t<u>If using Zombie NIR dye as live/dead discriminator</u>:\n\t\t<ul>\n\t\t\t<li>\n\t\t\t\tAdd 200 ml of PBS (RT) to all samples</li>\n\t\t\t<li>\n\t\t\t\tSpin at 2000 rpm for 1 minute 8&deg;C</li>\n\t\t\t<li>\n\t\t\t\tAdd 100 ml/well of Zombie Near-IR Live/Dead dye (1/2000) made up in PBS incubate at room temperature for 10 mins, add 200 ml <u>FACS buffer</u>.</li>\n\t\t</ul>\n\t</li>\n</ul>\n<p>\n\t<strong>General Recommendations for Setting up Cytometer</strong></p>\n<p style=\"margin-left:36pt;\">\n\tSet 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 of low frequencies (macrophages, DCs).</p>\n<p>\n\t<strong>Gating Panel 1</strong></p>\n<table border=\"1\" cellpadding=\"0\" cellspacing=\"0\" style=\"width:911px;\" width=\"683\">\n\t<colgroup>\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t</colgroup>\n\t<tbody>\n\t\t<tr height=\"33\">\n\t\t\t<td height=\"33\" style=\"height:44px;width:228px;\">\n\t\t\t\t<span style=\"font-size:12px;\">P<span style=\"font-family:arial,helvetica,sans-serif;\">arameters</span></span></td>\n\t\t\t<td colspan=\"7\" style=\"width:705px;\">\n\t\t\t\t<span style=\"font-size:12px;\"><span style=\"font-family:arial,helvetica,sans-serif;\">Gating steps</span></span></td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tPanel A live leukocyte count</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tT cells (panel A)</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNKT cells (panel A)</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNK cells (panel A)</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tOthers</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 T cells</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 T cells&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN T cells</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDP T cells</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 NKT cells</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 NKT cells&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN NKT cells</td>\n\t\t\t<td>\n\t\t\t\tnumber of live leukocytes</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD25+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD25- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD25+ T cells&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD25+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD25- T cells&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD25-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD25+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD25- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD25+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD25- NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD25+ NKT cells&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD25+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD25- NKT cells&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD25-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD25+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD25- NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD25-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD44+CD62L- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD44+CD62L+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD44-CD62L+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD44-CD62L- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD44+CD62L- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD44+CD62L+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD44-CD62L+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD44-CD62L- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD44+CD62L- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD44+CD62L+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD44-CD62L+ T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD44-CD62L- T cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD44+CD62L- NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD44+CD62L+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD4 CD44-CD62L+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4+</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD44+CD62L- NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD44+CD62L+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tCD8 CD44-CD62L+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8+</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD44+CD62L- NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD44+CD62L+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44+</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tDN CD44-CD62L+ NKT cells</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\tnumber of CD5+</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD4-</td>\n\t\t\t<td>\n\t\t\t\tCD8-</td>\n\t\t\t<td>\n\t\t\t\tCD44-</td>\n\t\t\t<td>\n\t\t\t\tCD62L+</td>\n\t\t</tr>\n\t</tbody>\n</table>\n<p>\n\t<strong>Gating Panel B</strong></p>\n<table border=\"1\" cellpadding=\"0\" cellspacing=\"0\" style=\"width:911px;\" width=\"683\">\n\t<colgroup>\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t\t<col />\n\t</colgroup>\n\t<tbody>\n\t\t<tr height=\"33\">\n\t\t\t<td height=\"33\" style=\"height:44px;width:205px;\">\n\t\t\t\t<span style=\"font-size:12px;\">Parameters</span></td>\n\t\t\t<td colspan=\"6\" style=\"width:705px;\">\n\t\t\t\t<span style=\"font-size:12px;\">Gating steps</span></td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tPanel B live leukocyte count</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNeutrophils</td>\n\t\t\t<td>\n\t\t\t\tLive</td>\n\t\t\t<td>\n\t\t\t\tCD11b+</td>\n\t\t\t<td>\n\t\t\t\tLy6G+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tMonocytes</td>\n\t\t\t<td>\n\t\t\t\tNot Granulocytes</td>\n\t\t\t<td>\n\t\t\t\tCD11b+</td>\n\t\t\t<td>\n\t\t\t\tLy6C High</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tEosinophils</td>\n\t\t\t<td>\n\t\t\t\tNot Monocytes</td>\n\t\t\t<td>\n\t\t\t\tCD11b+</td>\n\t\t\t<td>\n\t\t\t\tSSC-H High</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNK Cells (panel B)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNK Subsets (Q1)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD11b-</td>\n\t\t\t<td>\n\t\t\t\tLy6C+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNK Subsets&nbsp;(Q2)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD11b+</td>\n\t\t\t<td>\n\t\t\t\tLy6C+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNK Subsets&nbsp;(Q3)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD11b-</td>\n\t\t\t<td>\n\t\t\t\tLy6C-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNK Subsets&nbsp;(Q4)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD11b+</td>\n\t\t\t<td>\n\t\t\t\tLy6C-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNKT Cells (panel B)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNKT Subsets&nbsp;(Q1)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD11b-</td>\n\t\t\t<td>\n\t\t\t\tLy6C+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tNKT Subsets&nbsp;(Q3)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tCD11b-</td>\n\t\t\t<td>\n\t\t\t\tLy6C-</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tT Cells (panel B)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tT Subset</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tCD161-</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\tLy6C+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tB Cells</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tB1B Cells</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19+</td>\n\t\t\t<td>\n\t\t\t\tCD5+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tB2B Cells</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19+</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tFollicular B Cells</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19+</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD23+</td>\n\t\t\t<td>\n\t\t\t\tCD21/35+</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tpre-B Cells</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19+</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD23-</td>\n\t\t\t<td>\n\t\t\t\tCD21/35 low</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tMZB</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19+</td>\n\t\t\t<td>\n\t\t\t\tCD5-</td>\n\t\t\t<td>\n\t\t\t\tCD23 low</td>\n\t\t\t<td>\n\t\t\t\tCD21/35 High</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tcDCs</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD11c+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tcDCs CD11b Type</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD11c+</td>\n\t\t\t<td>\n\t\t\t\tCD11b+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tpDCs</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tNot T Cells</td>\n\t\t\t<td>\n\t\t\t\tLy6C+</td>\n\t\t\t<td>\n\t\t\t\tCD317+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tRP Macrophage (F4/80+)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tF4/80+</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tor</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t\t<tr>\n\t\t\t<td>\n\t\t\t\tRP Macrophage (CD19- CD11c-)</td>\n\t\t\t<td>\n\t\t\t\tNot Eosinophils</td>\n\t\t\t<td>\n\t\t\t\tMHCII+</td>\n\t\t\t<td>\n\t\t\t\tCD19-</td>\n\t\t\t<td>\n\t\t\t\tCD11c-</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t\t<td>\n\t\t\t\t&nbsp;</td>\n\t\t</tr>\n\t</tbody>\n</table>\n<p>\n\t&nbsp;</p>\n","Purpose":"<p>\n\tThis test differentiates immune cell sub-populations via flow cytometry.</p>\n<p>\n\t<strong>Description: </strong>increased CD4-positive T cell number (MP:0008074), decreased CD4-positive T cell number (MP:0008075), etc..</p>\n","Experimental Design":"<div class=\"expandable\">\n\t<ul>\n\t\t<li>\n\t\t\t<strong>Minimum number of animals :</strong>&nbsp; 3M + 3F</li>\n\t\t<li>\n\t\t\t<strong>Age at test: </strong>Week 16 </li>\n\t\t<li>\n\t\t\t<strong>Sex:</strong> We would expect the results of this test to show sexual dimorphism</li>\n\t</ul>\n</div>\n<p>\n\t&nbsp;</p>\n"}