Cd11b efluor 450 clone m1 70

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CD11b eFluor 450 (clone M1/70) is a fluorescent-labeled monoclonal antibody used for the detection and analysis of CD11b-expressing cells by flow cytometry. CD11b is a cell surface integrin expressed on various immune cells, including monocytes, macrophages, and granulocytes. This product provides a specific and reliable tool for the identification and characterization of CD11b-positive cells in research applications.

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Lab products found in correlation

Collagenase d, by Roche (4 mentions) Dnase 1, by Roche (3 mentions) Igm pe clone eb121 15f9, by BioLegend (2 mentions) Ly6g pe clone 1a8, by Thermo Fisher Scientific (2 mentions) Anti cd16 32 clone 93, by Thermo Fisher Scientific (2 mentions) C tube, by Miltenyi Biotec (2 mentions) Ack lysis buffer, by Thermo Fisher Scientific (2 mentions) Gentlemacs dissociator, by Miltenyi Biotec (2 mentions) Fortessa cytometer, by BD (2 mentions) Rneasy micro kit, by Qiagen (1 mentions) Flow jo fcs analysis software, by Tree Star (1 mentions) Siglec f pe cf594 clone e50 2440, by BD (1 mentions) Live dead aqua, by Thermo Fisher Scientific (1 mentions) Cd8 percp cy5 5 clone 53 6, by Thermo Fisher Scientific (1 mentions) Aqua live dead stain, by Thermo Fisher Scientific (1 mentions) F4 80 percp cy5, by Thermo Fisher Scientific (1 mentions) Ly6g pe, by Thermo Fisher Scientific (1 mentions) Cd4 fitc clone gk1, by BioLegend (1 mentions) Facsaria 3, by BD (1 mentions) Cd45 e780 clone30 f11, by Thermo Fisher Scientific (1 mentions)

7 protocols using cd11b efluor 450 clone m1 70

Multiparameter Flow Cytometry Analysis

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FACS was performed as described previously^{13 (link)}. Live cells were gated using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (cat. #L34957, Life Technologies, Grand Island, NY). The following antibodies were used for B cells: B220-PE-Cy7 (clone RA3-6B2, cat. #103221, Biolegend), and IgM-PE (clone eB121-15F9, cat. #12-5890-81, eBiosciences, San Diego, CA). The following antibodies were used for T cells and myeloid cells: CD4-FITC (clone GK1.5, cat. #11-0041-85; eBioscience), CD25–PE–Cy7 (clone PC61.5, cat. #25-0251-81; eBioscience), CD11b–eFluor 450 (clone M1/70, cat. #48-0112-82; eBioscience), and Ly6G-PE (clone 1A8, cat. #127607; BioLegend). RNA extraction of isolated cells for microarray analysis was performed using the RNEasy Microkit (Qiagen).

El-Zaatari M., Bass A.J., Bowlby R., Zhang M., Syu L.J., Yang Y., Grasberger H., Shreiner A., Tan B., Bishu S., Leung W.K., Todisco A., Kamada N., Cascalho M., Dlugosz A.A, & Kao J.Y. (2017). Indoleamine 2,3-dioxygenase 1, Increased in Human Gastric Pre-Neoplasia, Promotes Inflammation and Metaplasia in Mice and is Associated with Type II Hypersensitivity/Autoimmunity. Gastroenterology, 154(1), 140-153.e17.

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Isolation and Characterization of Murine Lung Mesenchymal Stem Cells

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The upper left lung lobe was removed and cut into small pieces with a razor blade. The lung tissue was then transferred to a C-tube (Miltenyi Biotec, Auburn, CA) and processed using digestion buffer containing 1mg/ml of Collagenase D and 0.1 mg/ml DNase I (Roche, Indianapolis, IN) in HBSS and a GentleMACS dissociator (Miltenyi Biotec), according to manufacturer’s instructions [28 (link)]. The homogenates were then filtered through 70 um nylon cell strainers to obtain a single cell suspension. Red blood cells were lysed using ACK lysis buffer (Life Technologies). Cells were counted using trypan blue to exclude dead cells. To assess mesenchymal stem cells, 1×10⁶ lung cells were first incubated with anti-CD16/32 (clone 93, eBioscience, San Diego, CA) to block unspecific binding to the Fcy II/III receptor. Cells were then incubated with rat anti-mouse F4/80 APC (clone BM8, eBioscience), CD11b eFluor 450 (clone M1/70, eBioscience), CD11c APC-eFluor 780 (clone N418, eBioscience), and Siglec-F PE-CF594 (clone E50-2440, BD Biosciences, San Jose, CA). Antibody incubation was carried out for 30 minutes at 4°C. Cells were washed and fixed as described [56 (link), 57 (link)]. Samples were run on a BD Fortessa cytometer (BD Biosciences). Data analysis was performed using Flow Jo FCS analysis software (Tree Star Inc., Ashland, OR).

Curtis B.J., Shults J.A., Boe D.M., Ramirez L, & Kovacs E.J. (2018). Mesenchymal Stem Cell Treatment Attenuates Liver and Lung Inflammation after Ethanol Intoxication and Burn Injury. Alcohol (Fayetteville, N.Y.), 80, 139-148.

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Multicolor Flow Cytometry Analysis of Gastric Immune Cells

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FACS was performed as described previously (10 (link)), using FACSAria III (BD, Franklin Lakes, NJ). Live cells were gated using LIVE/DEAD Aqua Stain (cat #L34957; Life Technologies, Grand Island, NY). For the quantification of immune cell frequencies within the gastric mucosae, the cells were stained with the following antibodies: (i) for myeloid cells: CD11b-eFluor 450 (clone M1/70, cat #48-0112-82; eBioscience, San Diego, CA) and Ly6G-PE (clone 1A8, cat. #127607; BioLegend, San Diego, CA); (ii) for T cells: CD4-FITC (clone GK1.5, cat. #11-0041-85, eBioscience) and CD8-PerCpCy5.5 (clone 53-6.7, cat. #45-0081, eBioscience); (iii) for Natural Killer cells: NK1.1-PECy5 (clone PK136, BioLegend); and (iv) for B cells: B220-PE-Cy7 (clone RA3-6B2, cat. #103221, BioLegend) and IgM-PE (clone eB121-15F9, cat. #12-5890, eBioscience).
For the characterization of gastric myeloid immune cell overlap using 7-color FACS, dissociated gastric cells were stained with: (i) LIVE/DEAD Aqua (cat #L34957; Life Technologies), (ii) CD11b-eFluor 450 (clone M1/70, cat #48-0112-82; eBioscience), (iii) Ly6G-PE (clone 1A8, cat. #127607; BioLegend), (iv) Ly6C-APC-Cy7 (clone HK1.4, cat #128025, BioLegend), (v) F4/80-PerCpCy5.5 (clone BM8, cat #45-4801-80, eBioscience), (vi) CD103-APC (clone 2E7, cat #17-1031-82, eBioscience), and (vii) CD11c-PeCy5 (clone N418, cat #15-0114-81, eBioscience).

Kao K.D., Grasberger H, & El-Zaatari M. (2023). The Cxcr2+ subset of the S100a8+ gastric granylocytic myeloid-derived suppressor cell population (G-MDSC) regulates gastric pathology. Frontiers in Immunology, 14, 1147695.

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Lung Immune Cell Isolation Protocol

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The upper left lung lobe was cut into small pieces, transferred to a C-tube (Miltenyi Biotec, Auburn, CA) containing digestion buffer (1mg/ml of Collagenase D and 0.1 mg/ml DNase I [Roche, Indianapolis, IN] in HBSS) and homogenized using a GentleMACS dissociator (Miltenyi Biotec), according to manufacturer guidelines. Single cell suspensions were obtained by passing homogenates through 70 um nylon cell strainers. Red blood cells were lysed in ACK lysis buffer (Life Technologies, Grand Island, NY) and remaining cells were counted using trypan blue exclusion of dead cells. Non-specific binding to the Fcy II/III receptor was prevented by incubating 1×10⁶ lung cells with anti-CD16/32 (clone 93,eBioscience, San Diego, CA). Cells were immunostained with rat anti-mouse antibodies: CD45 e780 (clone30-F11, eBioscience), CD11b eFluor 450 (clone M1/70, eBioscience), Ly6G (Gr-1) PE Cy7-conjugated (clone RB6-8C5, eBioscience), CD206 PE, CD24 APC, CD64 PerCP, and MHC II v500. Antibody incubation was carried out for 30 minutes at 4°C. Cells were washed and fixed as described (16 (link), 17 (link)). Flow experiments were performed using a BD Fortessa cytometer (BD Biosciences, San Jose, CA) and data analysed using Flow Jo FCS software (Tree Star Inc., Ashland, OR). Experiments were performed at minimum of two times, n=4–6 per group. Data from one representative experiment are shown.

Curtis B.J., Boe D.M., Shults J.A., Ramirez L, & Kovacs E.J. (2019). Effects of Multi-day Ethanol Intoxication on Post-Burn Inflammation, Lung Function, and Alveolar Macrophage Phenotype. Shock (Augusta, Ga.), 51(5), 625-633.

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Immune Cell Analysis of Olfactory Epithelium

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K18-hACE2 or BALB/c mice were deeply anesthetized with ketamine/xylazine OE isolated as described above. Isolated OE was digested with 1 mg/mL collagenase D (Roche) and 0.1 mg/mL DNase I (Roche) at 37°C for 20 minutes. Flow cytometric staining was performed on OE for immune cell analysis using the following antibodies: CD3-PE (clone 145-2C11; BioLegend), CD11b-eFluor 450 (clone M1/70; eBioscience), CD45-phycoerythrin-Cy7 (CD45-PE-Cy7) (clone 30-F11; BioLegend), CD4-PerCP-Cy5.5 (clone RM4-5; BioLegend), CD8-APC-Cy7 (clone 53–6.7; eBioscience), and Ly-6G-APC (neutrophil marker) (BioLegend). Cells were treated with anti-CD16/32, clone 2.4G2 generated in-house, to block nonspecific Fc receptor binding and were stained with the indicated antibodies at 4°C. Data were acquired with a BD FACSVerse cytometer and analyzed using FlowJo software (Tree Star Inc.).

Verma A.K., Zheng J., Meyerholz D.K, & Perlman S. (2022). SARS-CoV-2 infection of sustentacular cells disrupts olfactory signaling pathways. JCI Insight, 7(24), e160277.

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Lung Immune Cell Profiling Protocol

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To determine immune cell populations, lungs were digested (digestion buffer: 7 mL/sample Dulbecco’s modified Eagle’s medium + 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2% bovine serum albumin, 0.03 mg/mL Liberase Blendzyme 3 Collagenase Cocktail, 50 U/mL DNAse) and immune cells were isolated. Cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich), Ionomycin (1 μg/mL; Calbiochem), and brefeldin A. Cells were subsequently labeled with monoclonal antibodies. Specifically, Live/dead (Zombie UV Dye; BioLegend, San Diego, CA, USA), B220 – BV605 (clone RA3–6B2, BioLegend), CD45 – PEDazzle (clone 104, BioLegend), CD11b – eFluor450 (clone M1/70, eBioscience, San Diego, CA, USA), CD11c – BV711 (clone N418, BioLegend), NK1.1 – FITC (clone PK136, BioLegend), F4/80 – AF700 (clone BM8, eBioscience), Gr1 – APC (clone RB6–8C5, eBioscience), CD8 – BV510 (clone 53–6.7, BioLegend), and IL6 – PE (clone MP5–20F3, eBioscience). Data were collected using an LRS Fortessa flow cytometer (BD Biosciences) and analyzed by FlowJo X software (vX10, Tree Star, Ashland, OR, USA).

Alarcon P.C., Damen M.S., Ulanowicz C.J., Sawada K., Oates J.R., Toth A., Wayland J.L., Chung H., Stankiewicz T.E., Moreno-Fernandez M.E., Szabo S., Zacharias W.J, & Divanovic S. (2023). Obesity amplifies influenza virus-driven disease severity in male and female mice. Mucosal immunology, 16(6), 843-858.

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Skin Sample Analysis Protocol

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0.5‐cm² skin samples (from the central and lateral skin) were incubated with 2.5 mg/mL of Collagenase D (Roche Diagnostics) solution. Single‐cell suspensions from the skin were obtained by mashing the samples through 40‐μm cell strainers in RPMI1640 medium (Biowest) supplemented with 2% FBS (Gibco). The cells were stained for viability assessment (Zombie Aqua Fixable Viability Kit; BioLegend) and then blocked with anti‐CD16/CD32 antibodies (Fc block; eBioscience) followed by staining with directly conjugated antibodies: CD45.2‐APC/Cy7 (clone 104, BioLegend), CD11b‐eFluor450 (clone M1/70, eBioscience), Ly6G‐APC (clone 1A8, BioLegend) and CD3‐Alexa Fluor 594 (clone 17A2, BioLegend). Data were acquired on a BD LSRII (BD Biosciences). Singlets were selected based on FCS‐A vs FCS‐H. Dead cells were routinely excluded from the analysis. Analyses were performed with FCS Express (De Novo Software).

Kwiecinska P., Grygier B., Morytko A., Sanecka‐Duin A., Majchrzak‐Gorecka M., Kwitniewski M., Kapinska‐Mrowiecka M., Porebski G, & Cichy J. (2022). Secretory leukocyte protease inhibitor regulates nerve reflex‐mediated skin barrier function in psoriasis. Journal of the European Academy of Dermatology and Venereology, 36(8), 1266-1274.

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