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Anti cx3cr1

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Anti-CX3CR1 is a monoclonal antibody that binds to the CX3CR1 receptor, which is expressed on the surface of certain immune cells. CX3CR1 is involved in the migration and recruitment of these cells to sites of inflammation. The antibody can be used for the detection and analysis of CX3CR1-expressing cells in various applications, such as flow cytometry and immunohistochemistry.

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Lab products found in correlation

Anti ly6c, by BioLegend (2 mentions) Anti cd31, by BioLegend (2 mentions) Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions) Lamina propria dissociation kit, by Miltenyi Biotec (1 mentions) Anti cd45, by BioLegend (1 mentions) Zombie aqua, by BioLegend (1 mentions) Anti cd11b, by BioLegend (1 mentions) Anti ly6g, by BioLegend (1 mentions) Facsdiva software, by BD (1 mentions) Anti cd16, by BioLegend (1 mentions) Anti pd 1, by BioLegend (1 mentions) Anti tim 3, by BioLegend (1 mentions) Anti granzyme b, by BioLegend (1 mentions) Anti cxcr3, by BioLegend (1 mentions) Anti human lineage cocktail, by BioLegend (1 mentions) Anti cd27, by BioLegend (1 mentions) Anti tigit, by BioLegend (1 mentions) Anti nkp44, by BioLegend (1 mentions) Anti nkg2d, by BioLegend (1 mentions) Anti cd69, by BioLegend (1 mentions)

6 protocols using anti cx3cr1

1

Isolation and Analysis of Lung and Gut Immune Cells

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Mice were fasted overnight prior to the day of sacrifice. They were administered an overdose of isoflurane anesthesia, and perfused to remove blood prior to harvesting organs as described previously14 (link),28 (link),51 (link). Immune cells were isolated from lung by lung lavage21 (link) and from the lamina propria of the jejunum using a Miltenyi Biotech lamina propria dissociation kit (Catalog #130-097-410) as described28 (link). Single cell suspensions were incubated with Zombie Aqua (Biolegend, Catalog # 423101) together with anti-CD45 (Biolegend, catalog#103146), anti-CD11b (Biolegend, Catalog#101211), anti-Ly6C (Biolegend, Catalog#128037), anti-Ly6G (Biolegend, catalog#127639), anti-CX3CR1(Biolegend, Catalog#149015). After 45 min, the cells were washed twice with fluorescence-activated cell sorting (FACS) buffer (PBS + 5% FBS). After a short spin, the cells were suspended in 300 μL of ice-cold PBS buffer and transferred to fresh tubes for FACS analysis. FACS was performed using a BD LSR Fortessa X-20 machine SORP version 8.0.1 in the Janis V. Giorgi Flow Cytometry Core Facility at UCLA. For analysis and computational compensation of the data, BD FACS Diva software was used. Ten thousand events of live cells were gated. Only live and singlet cells were chosen for analysis and gating (i.e., dead cells and aggregates were excluded).
Chattopadhyay A., Yang X., Mukherjee P., Sulaiman D., Fogelman H.R., Grijalva V., Dubinett S., Wasler T.C., Paul M.K., Salehi-Rad R., Mack J.J., Iruela-Arispe M.L., Navab M., Fogelman A.M, & Reddy S.T. (2018). Treating the Intestine with Oral ApoA-I Mimetic Tg6F Reduces Tumor Burden in Mouse Models of Metastatic Lung Cancer. Scientific Reports, 8, 9032.
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2

Comprehensive NK Cell Phenotyping

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The following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.
Chen M., Li Y., Wu Y., Xie S., Ma J., Yue J., Lv R., Tian Z., Fang F, & Xiao W. (2021). Anti-Tumor Activity of Expanded PBMC-Derived NK Cells by Feeder-Free Protocol in Ovarian Cancer. Cancers, 13(22), 5866.
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3

Characterizing Monocyte Subsets by Flow Cytometry

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The expression of chemokine receptors was determined on the surface of fresh monocytes or cultured monocytes53 (link), 54 (link). Cells were incubated for 20 min at room temperature in the dark with anti-CD14, anti-CD163 (BDBioscience) anti-CD16 (Immunotools, Friesoythe, Germany), anti-CCR2 (R&D Systems, Weisbaden, Germany), anti-CCR5 (Clone:HEK/1/85a), anti-CXCR4, anti-CX3CR1, anti-CD206, and anti-HLA-DR (Biolegend, San Diego, CA) monoclonal-antibodies. Red blood cells were then lysed using RBC lysis buffer (1X) (Biolegend). Cells were washed twice with staining buffer (PBS, supplemented with 0.5% BSA; Calbiochem Merck, Darmstadt, Germany) and resuspended in 200 μl of staining buffer to be analyzed by flow cytometry.
The surface expression of different markers was analyzed on gated CD14+ monocytes and on gated monocyte subsets (identified as CD14+ CD16- (classical), CD14+ CD16+ (intermediate) and CD14lowCD16++ (non-classical)) using a MACSQuant® Instrument (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The percentage of positive cells (% cells) and mean fluorescence intensity (MFI) of each individual marker were calculated using © FlowJo, LLC 2013-2016 data analysis software.
Nieto J.C., Zamora C., Cantó E., Garcia-Planella E., Gordillo J., Ortiz M.A., Juárez C, & Vidal S. (2017). CSF-1 regulates the function of monocytes in Crohn’s disease patients in remission. Scientific Reports, 7, 92.
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4

Isolation and Characterization of Microglia

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Microglia cells were isolated through Percoll gradient or Miltenyi Adult Brain Dissociation kit, as described in RNA‐Seq Analysis. Microglia staining occurred in 1X dPBS−/− (Thermo Fischer) supplemented with 0.5% FBS, 0.4% 0.5 M EDTA, and 1% HEPES at 4°C for 20 min. Cells were stained with the following antibodies: anti‐CD11b (clone:M1/70, eBioscience), anti‐CD45 (clone:30‐F11, eBioscience), anti‐F480 (clone:BM8, eBiosience), anti‐CX3CR1 (clone:SA011F11, Biolegend), anti‐CD31 (clone:MEC13.3, Biolegend), anti‐CCR3 (clone:J073E5, Biolegend), anti‐ I‐A/I‐E (clone:M5/114.15.2, Biolegend), anti‐CD80 (clone:16‐10A1, eBioscience), anti‐CD86 (clone:GL1, eBioscience), and anti‐TLR4 (clone:SA15‐21, Biolegend). Following staining, cells were washed twice and fixed in a 1:1 solution of supplemented dPBS−/−: 4% paraformaldehyde overnight at 4°C. After fixation, cells were resuspended in supplemented dPBS−/−and enumerated via flow cytometry (BD LSR II with 561 laser). Microglia populations were identified as CD11bhigh/CD45low. Subsequent data was analyzed using FlowJo software (v. 10.5.3).
Bauer K.C., York E.M., Cirstea M.S., Radisavljevic N., Petersen C., Huus K.E., Brown E.M., Bozorgmehr T., Berdún R., Bernier L., Lee A.H., Woodward S.E., Krekhno Z., Han J., Hancock R.E., Ayala V., MacVicar B.A, & Finlay B.B. (2022). Gut microbes shape microglia and cognitive function during malnutrition. Glia, 70(5), 820-841.
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5

Immune Cell Phenotyping via Flow Cytometry

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anti-Cd11b [M1/70] PE (Biolegend, 101208, 1:200), anti-Cd45 APC-Cy7 (Biolegend, 103116, 1:200), anti-Cx3cr1 (Biolegend, 149008, 1:200).
Hammond T.R., Dufort C., Dissing-Olesen L., Giera S., Young A., Wysoker A., Walker A.J., Gergits F., Segel M., Nemesh J., Marsh S.E., Saunders A., Macosko E., Ginhoux F., Chen J., Franklin R.J., Piao X., McCarroll S.A, & Stevens B. (2018). Single cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes. Immunity, 50(1), 253-271.e6.
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6

Comprehensive Multiparameter Flow Cytometry

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Flow cytometry was performed using the BD FACSCanto II and analysed with FlowJo software (Tree Star, Ashland, OR). Dead cells were excluded through Fixable Viability Dye eFluor staining (eBioscience). Nonspecific antibody binding was blocked with an anti-CD16/32 (Mouse Fcγ block clone 2.4G2). The cells were incubated for 30 min in 100 μl PBS with conjugated antibodies (BD Biosciences, France): anti CD45 (V500 conjugated; clone 30-F11), CD11b (BV421 conjugated; clone M1/70), anti CD3 (FITC conjugated; clone 145-2C11), anti CD45R/B220 (PE-Cy7 conjugated; clone RA3-6B2), anti Ly6C (FITC conjugated; clone AL-21), anti CD31 (PE conjugated; clone MEC13.3), anti CD34 (Alexa Fluor 647 conjugated; clone RAM34), anti CD146 (FITC conjugated; clone ME-9F1), anti CD29 (PE conjugated; clone HM B1.1), anti Sca-1 (PE-Cy7 conjugated; clone D7), anti CD44 (PE-Cy5.5 conjugated; clone IM7), anti CD105 (Alexa Fluor 647 conjugated; clone MJ7/18), anti SSEA (BV421 conjugated; clone MC631), anti CX3CR1 from Biolegend (France) (PE-Cy7 conjugated; clone SA011F11), anti CCR2 (Alexa Fluor 647 conjugated; clone SA203G11) and CD90 from southern Biotech (FITC conjugated; clone SA203G11). Isotype-matched antibodies (BD Biosciences) were used for control staining. All antibodies were used at 1:100 dilution. The concentration of cell suspensions was adjusted to 1 × 106 cells per 100 μl.
Bensemmane L., Squiban C., Demarquay C., Mathieu N., Benderitter M., Le Guen B., Milliat F, & Linard C. (2021). The stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome in mice. Stem Cell Research & Therapy, 12, 309.
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