A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment

A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment
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  A Novel Ly6C/Ly6G-Based Strategy to Analyzethe Mouse Splenic Myeloid Compartment Shawn Rose, 1,2 Alexander Misharin, 1,2 Harris Perlman 1,2 *  Abstract Currently, there is no standardized panel for immunophenotyping myeloid cells inmouse spleen using flow cytometry. Markers such as CD11b, CD11c, F4/80, Gr-1,Ly6C, and Ly6G have long been used to identify various splenic cell myeloid popula-tions. Flow cytometry and fluorescence-activated cell sorting (FACS) analysis demon-strated that Ly6G/Ly6C markers are superior to Gr-1 for identifying splenic neutrophils,eosinophils, and subsets of monocytes/macrophages. Moreover, these experimentsshowed that F4/80 is not required for identifying these myeloid subsets and that many of the commercially available preparations of anti-F4/80 antibodies stain poorly for thisantigen in spleen. Taken together, we have now developed an informative flow cytome-try panel that can be combined with other cell markers to further delineate subpopula-tions of mouse splenic myeloid cells. This panel will be highly useful to investigators inthe flow cytometry field, as there is a critical need to standardize the analysis of myeloidcell subsets.  '  2011 International Society for Advancement of Cytometry   Key terms macrophage; monocyte; F4/80; immunophenotyping I NTRODUCTION The mouse spleen contains several distinct populations of myeloid cells withvarying immune functions, including neutrophils, eosinophils, monocytes, macro-phages, and dendritic cells. Although previous studies have suggested that these cellsare readily identified by flow cytometry based on their cell surface staining character-istics, many of these cells share common expression patterns for myeloid specificantigens. Therefore, a single antibody is not sufficient for demarcation of various my-eloid subsets and a need for standardized markers to phenotype mouse myeloid cellsin the spleen is required, which is particularly true for monocytes/macrophages.Under steady-state conditions, most macrophage populations within murinelymphoid tissues are believed to srcinate from blood monocytes. On the basis of theexpression of cell surface markers, mouse monocytes can be divided into two mainsubsets: classical (Ly6C 11 CD43CCR2 1 CD62L 1 CX3CR1 Low  ) and nonclassical(Ly6C 2 CD43 1 CCR2 2 CD62L 2 CX3CR1 Hi ) (1). Mouse macrophages have also typically been divided into two subsets based on the expression of Gr-1 or Ly6C antigens. TheLy6C (or Gr-1) Hi subset has been termed ‘‘classical’’ or ‘‘inflammatory’’ while Ly6C (orGr-1) Low-neg cells are termed ‘‘nonclassical’’ or ‘‘resident’’ (2,3). Both of these subpopu-lations express the 125 kDa transmembrane adhesion glycoprotein F4/80, (4) which isnot essential for macrophage function (5). Antibodies to the F4/80 antigen were srcin-ally derived by fusing splenocytes from a rat hyperimmunized with cultured thioglycol-late-induced mouse peritoneal macrophages with a mouse myeloma cell line (4). It hasgenerally been assumed to be a macrophage-specific marker, yet other cell types, suchas skin Langerhans cells (6) and eosinophils (7), also express F4/80.To achieve a panel for immunophenotyping splenic myeloid cells, various cellsurface markers were tested by flow cytometry and fluorescence-activated cell sorting 1 Department of Medicine, NorthwesternUniversity Feinberg School of Medicine,Chicago, Illinois 60611 2 Division of Rheumatology, NorthwesternUniversity Feinberg School of Medicine,Chicago, Illinois 60611Received 5 August 2011; RevisionReceived 14 October 2011; Accepted 8December 2011Additional Supporting Information may befound in the online version of this article.Shawn Rose and Alexander Misharincontributed equally to this study.Grant sponsors: NIH Loan RepaymentGrant; Grant sponsor: NIH/NIAMS T32;Grant number: AR07611; Grant sponsor:NIH/NIAID R21; Grant number: AI092490;Grant sponsor: NIH/NIAMSR01; Grantnumbers: AR050250, AR054796.*Correspondence to: Harris Perlman,Departments of Medicine andRheumatology, Northwestern UniversityFeinberg School of Medicine, 240 EastHuron Street, Room M338, Chicago, IL60611E-mail: h-perlman@.northwestern.eduPublished online in Wiley Online Library( 10.1002/cyto.a.22012© 2011 International Society forAdvancement of Cytometry TechnicalNote Cytometry Part A    00A: 000  000, 2011  (FACS) analysis. Compared with Gr-1, Ly6C/Ly6G markerswere better for identifying neutrophils, eosinophils, and bothsubsets of monocytes/macrophages in mouse spleen. Detailedinvestigations using the antigen F4/80 revealed that myeloidcell subsets could be readily identified without the use of thismarker. Furthermore, many of the commercially availableanti-F4/80 antibodies stained weakly for this antigen. Herein,a splenic myeloid cell immunophenotyping panel that can beused independently or in combination with other markers isprovided. Adoption of this improved panel will be imperativefor standardizing the investigation of myeloid cell subsets inmouse spleen. M ATERIALS AND M ETHODS Animals C57BL/6 (B6) mice and B6.129P- Cx3cr1 tm1Litt  / J (furtherreferred to as CX3CR1-GFP/GFP) mice were initially obtainedfrom the Jackson Laboratory (Bar Harbor, ME). Only CX3CR1-GFP/ 1  animals were used in our studies. Animalswere bred and maintained in a pathogen free barrier facility within the Center for Comparative Medicine at NorthwesternUniversity. All experiments involving mice were approved by the IACUC at Northwestern University. Cell Preparation Spleens were harvested and pooled from 6 to 8 week oldB6 or CX3CR1-GFP/ 1 mice in RPMI 1640 (Mediatech, Man-assus, VA). Cell suspensions were prepared by dicing spleenswith a razor blade, digesting with a solution containing0.1 mg/mL DNase I (Roche, Indianapolis, IN) and 1 mg/mLCollagenase D (Roche, Indianapolis, IN) in HBSS (Cellgro,Manassus, VA) for 30 min at 37 8 C, followed by passagethrough a 40  l M Nylon filter (BD Falcon, Bedford, MA). Redblood cell lysis was performed using 2 mL/spleen of 1x BDPharm Lyse solution (BD Biosciences, Sparks, MD). Cells werethen washed in either MACS buffer (Miltenyi Biotech,Auburn, CA) or staining buffer (Ca 2 1 and Mg 2 1 free PBS(BioWhittaker, Wakersville, MD) containing 5% heat-inacti-vated fetal bovine serum (Atlas, Fort Collins, CO), 0.09% so-dium azide (Sigma-Aldrich, St. Louis, MO), and 5 mM EDTA(Acros Organics, Geel, Belgium) and counted using a Coun-tess automated cell counter (Invitrogen, Carlsbad, CA). Forflow cytometry immunophenotyping experiments, 3  3  10 6 cells per tube were stained as described below. For FACS analy-ses, 2  3  10 8 cells per cocktail were prepared in MACS buffer(Miltenyi Biotech), rather than staining buffer. Flow Cytometric Cell Staining Cell viability was assessed by incubation in the amine-re-active dye Aqua [(Invitrogen) (1:500) dilution in Ca 2 1 andMg 2 1 free PBS)] for 30 min in the dark at room temperature(RT), followed by a single wash in 1x PBS. For all experiments,cells were incubated in 0.5  l g Fc Block (BD Biosciences) for10 min at RT. Surface staining was performed in the dark for30 min at 4 8 C in staining buffer. Cells were then washed twicewith staining buffer followed by fixation in 1% paraformalde-hyde (VWR, West Chester, PA). A comprehensive list of sur-face markers for these experiments includes: CD45R (B220)clone RA3-6B2 PE-Texas Red (1:250, BD Biosciences), CD4clone RM4-5 PerCP-Cy5.5 (1:160, BD Biosciences), CD8 clone53-6.7 PerCP-Cy5.5 (1:160, BD Biosciences), CD8 clone 53-6.7 eFluor 450 (1:333, eBioscience), CD11b clone M1/70eFluor 450 (1:160, eBioscience, San Diego, CA), CD11b cloneM1/70 PE-Texas Red [1:500, Invitrogen (Caltag)], CD11cclone HL3 PE-Cy7 (1:125, BD Biosciences), CD16/CD32 clone2.4G2 PE (1:100, BD Biosciences), CD19 clone 1D3 PerCP-Cy5.5 (1:160, BD Biosciences), CD40 clone 1C10 APC (1:100,eBioscience), CD69 clone H1.2F3 PerCP-Cy5.5 (1:167, BDBiosciences), CD80 clone 16-10A1 PE (1:500, eBioscience),CD86 clone GL1 APC (1:333, eBioscience), CD86 clone GL1Alexa Fluor 700 (1:100, BD Biosciences), CD115 APC cloneAFS98 (1:100, eBioscience), F4/80 clone CI:A3-1 Alexa Fluor647 (various dilutions, optimized at 1:200, AbD Serotec, Ra-leigh, NC), F4/80 clone BM8 Alexa Fluor 700 (various dilu-tions, AbD Serotec), F4/80 clone BM8 APC (various dilutions,optimized at 1:100, eBioscience), F4/80 clone BM8 FITC (vari-ous dilutions, eBioscience), F4/80 clone BM8 PE (various dilu-tions, eBioscience), F4/80 clone BM8 PE-Cy7 (various dilu-tions, eBioscience), F4/80 clone BM8 PE-Texas Red (variousdilutions, eBioscience), F4/80 clone BM8 PerCP-Cy5.5 (vari-ous dilutions, eBioscience), Gr-1 clone RB6-8C5 APC-Cy7(1:160, BD Biosciences), Ly6C clone AL-21 APC-Cy7 (1:500,BD Biosciences), Ly6G clone 1A8 PE (1:416, BD Biosciences),Ly6G clone 1A8 PerCP-Cy5.5 (1:500, BD Biosciences), MHCClass II clone M5/114.15.2 FITC (1:167, eBioscience), MHCClass II clone M5/114.15.2 eFluor 450 (1:500, eBioscience),MHC Class II clone M5/114.15.2 APC-eFluor 780 (1:167,eBioscience), NK1.1 clone PK136 Alexa Fluor 700 (1:160, BDBiosciences), NK1.1 clone PK136 APC (1:286, BD Bios-ciences), NK1.1 clone PK136 FITC (1:160, BD Biosciences),NK1.1 clone PK136 PerCP-Cy5.5 (1:100, BD Biosciences),mPDCA-1 clone JF05-1C2.4.1 APC (1:10, Miltenyi Biotech),Siglec F clone E50-2440 PE (1:100, BD Biosciences). For flow cytometry immunophenotyping experiments, cells wereacquired on an LSR II cytometer (BD Immunocytometry Systems, San Jose, CA) equipped with 405 nm, 488 nm, 561nm, and 640 nm excitation lasers. The spleen FACS experi-ments were performed using a FACSAria II instrument (BDImmunocytometry Systems) equipped with 405 nm, 488 nm,or 633 nm lasers located at the University of Chicago Flow Cytometry Core Facility, Chicago, IL. All data collection andsorting were performed using BD FACS Diva software (BDBiosciences) and data analyses were performed using FlowJosoftware (Tree Star, Ashland, OR). Fluorescence minus one(FMO) controls were used for gating analyses to distinguishpositively from negatively staining cell populations. Com-pensation was performed using single color controls pre-pared from BD Comp Beads (BD Biosciences) for cell surfacestaining or Arc Beads (Invitrogen) for Aqua live/dead discri-mination. Compensation matrices were calculated andapplied using FlowJo software (Tree Star). Biexponentialtransformation was adjusted manually when necessary. Forconfiguration of the BD FACSAria III at the University of  TECHNICAL NOTE 2  Ly6C and Ly6G Reliably Identify Murine Splenic Myeloid Cells   Chicago Flow Cytometry core facility, please contact RyanDuggan ( Cytospin Preparation and Staining For cytologic analysis of cell preparations, cells weremounted on slides using a Cytospin centrifuge (Shandon, Ram-sey, MN) for 10 min at 1,000 RPM. Cells were then fixed inmethanol for 30 s and air-dried. Slides were then submerged inDiff-quick solution II for 30 s and drained, followed by immer-sion in Diff-quick solution I for 30 s. The slides were rinsed intap water for 60 s and then rapidly dehydrated in absolute alco-hol. Counterstaining with Giemsa was performed for 30 s, fol-lowed by rinsing with tap water and rapid dehydration in abso-lute ethanol. Cytospin preparations were imaged using anOlympus (Tokyo, Japan) microscope equipped with an Olym-pus DP20 imaging system. Independent analysis of the slideswas performed by a hematopathologist blinded to the study. R ESULTS F4/80 Staining Is Highly Variable in Mouse Spleen F4/80 has long been used to identify monocytes and mac-rophages in both lymphoid and nonlymphoid tissues. To opti-mize F4/80 staining by flow cytometry in mouse spleen, wetested the commonly used clone BM8 conjugated to variousfluorochromes (Supporting Information Fig. 1). F4/80 stain-ing was highly variable, with several different fluorochromesconjugated to BM8 giving high background signal that pre-vented clear delineation of positively and negatively stainedcell populations. This was particularly true in the FMO panels.The clone CI:A3-1 conjugated to Alexa Fluor 647 gave the bestoverall signal-to-noise ratio, allowing for clear distinction of F4/80 positive and negative cells irrespective of CD11b expres-sion (Supporting Information Fig. 1).Upon building a splenic phenotyping panel, it becameapparent that combinations of the BM8 clone with Gr-1resulted in poor discrimination of F4/80 positive and negativepopulations for CD11b Hi cells (Supporting Information Fig. 2,Gates C and E). This effect was also evident, although less ro-bust, when using the CI:A3-1 clone (Supporting InformationFig. 3, Gates C and E). Combining either F4/80 clone with var-ious other targets including Ly6C, Ly6G, NK1.1, and CD11cdid not reproduce this effect (Supporting Information Figs. 2and 3 and data not shown). The inability to discriminate F4/80 positive and negative populations may not be a compensa-tion issue, as excitation of various other antigenic targets usingthe 640 nM laser did not produce a similar result (SupportingInformation Figs. 2 and 3 and data not shown). Preincubationwith Gr-1 prior to staining with either F4/80 clone correctedthis issue, while the converse experiment completely maskedthe CD11b 1 F4/80 2 cell population (Supporting InformationFig. 4, Gate E and data not shown). Notably, preincubationwith either F4/80 clone prior to Ly6G staining also obscuredthe CD11b 1 F4/80 2 population. Taken together, these findingssuggest that commercially available preparations of anti-F4/80antibodies vary considerably in their ability to identify F4/80positive cells in mouse spleen. Another possibility is that theremay be a common epitope between F4/80 and the Ly6G com-ponent of Gr-1 or steric hindrance between the antibodies, asevidenced by the competition experiments. Gr-1 and Ly6C/Ly6G Markers Identify SimilarPopulations of Monocytes and Macrophagesin Mouse Spleen Given the above findings, we set out to establish a gatingstrategy for phenotyping mouse myeloid cells in spleen with-out examining the F4/80 antigen (Fig. 1). After exclusion of debris, doublets, nonviable cells, and lineage markers (CD4,CD8, CD19, NK1.1, CD11c Hi ), CD11b 1 cells were subdividedinto 4 distinct populations using either Gr-1 vs SSC or a com-bination of Ly6C and Ly6G vs SSC markers (Fig. 1): (A) neu-trophils, Ly6G Hi SSC Int or Gr-1 Hi SSC Int , (B) eosinophils,Ly6C Int Ly6G 2 SSC Hi or Gr-1 Int SSC Hi , and monocytes andmacrophages, (C) Ly6C Lo-neg Ly6G 2 SSC Lo or Gr-1 Lo-neg SSC Lo or (D) Ly6C 1 Ly6G 2 SSC Lo or Gr-1 1 SSC Lo . The majority of Ly6C Lo-neg Ly6G 2 SSC Lo or Gr-1 Lo-neg SSC Lo cells were MHCClass II 1 CD115 2 , consistent with a macrophage phenotype[(8) and Fig. 1]. In contrast, Ly6C 1 Ly6G 2 SSC Lo or Gr-1 1 SSC Lo cells were predominantly MHC Class II 2 CD115 1 ,suggestive of a classical monocyte phenotype [(9) and Fig. 1].Neutrophils and eosinophils were negative for MHC Class IIand CD115 staining (Supporting Information Fig. 5). Identifi-cation of the Ly6C Int Ly6G 2 SSC Hi eosinophil population wasfurther confirmed by Siglec F staining (Fig. 2).Previous studies have utilized anti-Gr-1 antibodies or acombination of anti-Ly6C/anti-Ly6G antibody staining tocharacterize myeloid cell populations in mouse spleen. Directcomparison of sorted populations using Gr-1 or Ly6C/Ly6Gsurface markers revealed that Gr-1 Hi SSC Int cells included neu-trophils, monocytes, and macrophages (Supporting Informa-tion Fig. 6A) while Ly6G Hi SSC Int cells were entirely neutro-phils and Ly6C Lo-neg Ly6G 2 SSC Lo and Ly6C 1 Ly6G 2 SSC Lo cellswere monocytes and macrophages (Supporting InformationFig. 6B). Because anti-Gr-1 antibody recognizes both Ly6Cand Ly6G antigens (10,11) and differences in intensity of thestaining between neutrophils and classic monocytes/macro-phages may not be sufficient for their accurate separation, ourdata suggest the use of anti-Ly6C/anti-Ly6G antibodies fordelineating splenic myeloid cells populations. Staining for the F4/80 Antigen Is Not Required toIdentify Myeloid Cell Subsets in Mouse Spleen The requirement of anti-F4/80 antibody for flow cytometricimmunophenotyping of monocytes and macrophages in spleenis not known. To directly test this hypothesis, splenocytes weresorted followed by cytospin preparation and staining with Diff-quick and Giemsa (Fig. 3). Samples were stained concurrently with two different cocktails. The first cocktail contained CD11b,Ly6C, Ly6G, CD11c, and F4/80 (Fig. 3a) while the second cock-tail contained CD11b, Ly6C, Ly6G, CD11c, and NK1.1 (Fig. 3b).After gating out debris, doublets, and nonviable cells, four sub-populations of CD11b 1 cells were sorted. Both sorts yieldedidentical results independent of F4/80 being included in thecocktail. Ly6G Hi SSC Int cells were exclusively neutrophils (Figs. 3a TECHNICAL NOTE Cytometry Part A    00A: 1  8, 2011  3  and 3b, Gate A). The Ly6C Int Ly6G 2 SSC Hi population consistedalmost entirely of eosinophils with very rare monocytes/macro-phages (Figs. 3a and 3b, Gate B). Ly6C Lo-neg Ly6G 2 SSCLo andLy6C 1 Ly6G 2 SSC Lo cells were all monocytes and macrophages.Taken together, these data indicate that F4/80 is not required foridentifying mouse splenic myeloid cell sub-populations. Distinguishing Monocytes from Macrophages inMouse Spleen Swirski et al. (9) have previously shown that the mousespleen serves as a reservoir for monocytes that can be mobilizedin response to inflammatory signals. These investigators definedmonocytes as Lin 2 CD11b Hi CD11c Lo MHC Class II 2 F4/80 Lo cells, which were further split into Ly6C Lo and Ly6C Hi subpopu-lations. To address whether splenic monocytes and macro-phages can reliably be distinguished based on expression of cellsurface markers, we performed flow cytometric immunopheno-typing on CX3CR1-GFP/ 1 mice (12). After exclusion of debris,doublets, dead cells, NK cells, dendritic cells, neutrophils, andeosinophils, CD11b 1 cells were divided into CD11b 1 F4/80 2 orCD11b 1 F4/80 1 populations. Each of these subsets was thenfurther segregated into Ly6C Lo-neg SSC Lo and Ly6C Hi SSC Lo populations (Supporting Information Fig. 7). In both the F4/80 2 and F4/80 1 populations, the majority of CD11b 1 Ly6C Lo-neg cells were MHC Class II 1 CD115 2 while CD11b 1 Ly6C Hi cells were MHC Class II 2 CD115 1 . CX3CR1 expression, asmeasured by GFP fluorescence, was higher among CD11b 1 F4/80 1 Ly6C Lo-neg cells compared with CD11b 1 F4/80 2 Ly6C Lo-neg cells. In contrast, levels of CX3CR1 were similar inCD11b 1 F480 1 Ly6C Hi and CD11b 1 F480 2 Ly6C Hi cells.Since the markers used thus far could not reliably identify splenic monocytes, we further phenotyped Ly6C Lo-neg and Figure 1.  Gr-1 and Ly6C/Ly6G markers identify similar populations of monocytes and macrophages in the mouse spleen. Total mouse sple-nocytes from C57BL/6 mice were prepared as described in Materials and Methods. Debris (SSC-A vs. FSC-A) and doublets (FSC-H vs. FSC-A)were excluded and live/dead discrimination was determined using the amine reactive dye Aqua (FSC-H vs. Pacific Orange-A Live/Dead).CD11b 1 Lineage 2 cells (gated out using a CD4, CD8, CD19 PerCP-Cy5.5 dump channel) were then sub-gated on CD11b 1 NK1.1 2 cells, followedby excluding CD11c Hi cells. For the first cocktail (CK1), four subpopulations were identified: ( A ) Gr-1 Hi SSC Int cells (neutrophils) and ( B ) Gr-1 Lo-neg SSC Hi cells (eosinophils) were negative for MHC Class II and CD115 staining (Supporting Information Fig. 5) ( C ) Gr-1 Lo-neg SSC Lo cells(monocytes/macrophages) were MHC Class II 1  /  2 CD115 2 (red histogram) ( D ) Gr-1 1 SSC Lo cells (monocytes/macrophages) were MHC ClassII 2 CD115 1  /  2 (blue histogram). For the second cocktail (CK2), four subpopulations were identified: (A) Ly6G Hi CD11b 1 cells (neutrophils) and(B) Ly6C Lo-neg Ly6G 2 SSC Hi cells (eosinophils) were negative for MHC Class II and CD115 (Supporting Information Fig. 5) (C) Ly6C Lo-neg Ly6G 2 SSC Lo cells (monocytes/macrophages) were MHCII 1  /  2 CD115 2 (red histogram) (D) Ly6C 1 Ly6G 2 SSC Lo cells (monocytes/macro-phages) were MHC Class II 2 CD115 1  /  2 (blue histogram). Frequencies of cells in each sub-gate (after debris and doublet exclusion) areexpressed as a percentage of live cells. TECHNICAL NOTE 4  Ly6C and Ly6G Reliably Identify Murine Splenic Myeloid Cells   Ly6C Hi monocytes/macrophages for various cell surface anti-gens (Fig. 4). The activation markers CD40, CD80, and CD86were all more highly expressed on Ly6C Lo-neg cells comparedwith Ly6C Hi cells. Ly6C Lo-neg cells also expressed greater levelsof CX3CR1 and MHC Class II, heterogeneous levels of Fc c R,and lower levels of CD115 relative to their Ly6C Hi counter-parts. Together, these data suggest that expression of F4/80cannot be used to discriminate between mouse splenic mono-cytes and macrophages. In contrast, Ly6C Lo-neg and Ly6C Hi monocytes/macrophages differentially express various pheno-typic markers, which could be indicative of distinct matura-tion stages between these cell populations. Immunophenotyping Dendritic Cell Populations inMouse Spleen To expand the analysis of splenic myeloid cells, we incor-porated the markers CD8 a  and mPDCA-1 into our panel toidentify subsets of dendritic cells in CX3CR1-GFP/ 1  mice(Supporting Information Fig. 8). After exclusion of debris,singlets, and nonviable cells, splenocytes were gated intoB220 1 and B220 2 populations. B220 1 CD11c Int mPDCA-1 1 plasmacytoid dendritic cells (pDC) and B220 2 CD11c Hi CD8 2 and B220 2 CD11c Hi CD8 1 conventional dendritic cells (cDC)were easily identified. Further analysis revealed that both sub-sets of cDC expressed higher amounts of CD80 and MHCClass II and lower levels of CD69 compared to pDC. CX3CR1expression was found to be greatest in CD8 2 cDC. High levelsof CX3CR1 expression was also detected in a small subpopula-tion of CD8 1 cDC, which are believed to be more closely related to pDC (13). Therefore, a minimal panel of core mar-kers including B220, CD11c, mPDCA-1, and CD8 a  can beused to phenotype DC populations in mouse spleen. D ISCUSSION F4/80 has generally been considered to be a marker speci-fic for both circulating monocytes and tissue-residing mono-cytes and macrophages, yet its expression is not limited tothese cell populations (6,7). Here, we have demonstrated that Figure 2.  Gr-1 Lo-neg SSC Hi mouse splenocytes are eosinophils. As in Figure 1, debris, doublets, and nonviable cells were excluded fromtotal C57BL/6 mouse splenocytes. Live cells were sub-gated on CD11b 1 Lineage 2 cells (gated out using a CD4, CD8, CD19, NK1.1 PerCP-Cy5.5 dump channel). After gating out CD11c Hi cells, four subpopulations of cells were identified: ( A ) Gr-1 Hi SSC Int cells (neutrophils) and( B ) Gr-1 Lo-neg SSC Hi cells (eosinophils) were positive for Siglec F staining on sub-gate analysis, ( C ) Gr-1 Lo-neg SSC Lo and ( D ) Gr-1 1 SSC Lo monocytes/macrophages were also identified. Gate E, which encompasses gates A, C, and D, was negative for Siglec F staining. Frequen-cies of cells in each sub-gate (after debris and doublet exclusion) are expressed as a percentage of live cells. TECHNICAL NOTE Cytometry Part A    00A: 1  8, 2011  5
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