Bone marrow (BM) cells were isolated by flushing long bones with PBS and filtering through a 70-μm cell strainer (BD, Inc. Cat.#352350). After red blood cell lysis, BM cells were analysed using PE-conjugated CD11b (BD, Inc. Cat.#553311), FITC-conjugated Gr-1 (eBioscience, Inc. Cat.#RB6-8C5) and APC-conjugated Ly6C (BD, Inc. Cat.#557661) antibodies with Gallios flow cytometer (Beckman Coulter, Inc. Brea, CA, USA).
Pe conjugated cd11b
Manufactured by BD
Sourced in United States
PE-conjugated CD11b is a cell surface protein marker used for flow cytometry applications. It is conjugated to the fluorescent dye phycoerythrin (PE) for detection. CD11b is expressed on the surface of various immune cells, including monocytes, macrophages, and granulocytes. This marker can be used to identify and characterize these cell populations.
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Lab products found in correlation
Fitc conjugated gr 1, by Thermo Fisher Scientific (1 mentions)
Apc conjugated ly6c, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Rnase a, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Percpcy5.5 conjugated f4 80, by BioLegend (1 mentions)
Pe cy7 conjugated cd3, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated cd14, by BioLegend (1 mentions)
Fcr blocker, by BD (1 mentions)
Facsverse system, by BD (1 mentions)
Creative suite, by Adobe (1 mentions)
Fitc conjugated anti f4 80, by Thermo Fisher Scientific (1 mentions)
Pe cy7 conjugated anti cd11c, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by Tree Star (1 mentions)
Apc conjugated annexin 5, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Calibur flow cytometer, by BD (1 mentions)
7 protocols using pe conjugated cd11b
1
Bone Marrow Cell Profiling
2
Cell Proliferation and Cell Cycle Analysis
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Cell proliferation was measured by CCK-8 (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s instructions. The optical absorbance was measured at 450 nm by a plate reader (BioTek Instruments, Inc., Winooski, VT, USA).
Antibodies PE-conjugated CD11b were purchased from BD PharMingen (CA, USA). Total cells were Fcblocked and stained with indicated combinations of antibodies for 30 min on ice, then washed three times and resuspended in 1% FBS/PBS. For cell cycle assays, total cells were harvested and washed three times in PBS (pH 7.4), followed by fixation with precooled 70% ethanol overnight at 4 °C. Ethanol-fixed cells were centrifuged at 1000 rpm for 5 min, washed twice with PBS, and then incubated with 0.5 mL PBS containing 10 g/mL RNase A and 50 g/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark at 4 °C. The flow cytometric data were collected on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software or ModFit LT3.0 software.
Antibodies PE-conjugated CD11b were purchased from BD PharMingen (CA, USA). Total cells were Fcblocked and stained with indicated combinations of antibodies for 30 min on ice, then washed three times and resuspended in 1% FBS/PBS. For cell cycle assays, total cells were harvested and washed three times in PBS (pH 7.4), followed by fixation with precooled 70% ethanol overnight at 4 °C. Ethanol-fixed cells were centrifuged at 1000 rpm for 5 min, washed twice with PBS, and then incubated with 0.5 mL PBS containing 10 g/mL RNase A and 50 g/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark at 4 °C. The flow cytometric data were collected on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software or ModFit LT3.0 software.
3
Multiparameter Flow Cytometry Profiling
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The following fluorochrome-conjugated monoclonal antibodies were used in this study: anti-mouse PE/Cy7-conjugated CD45 (Cat# 940335), APC-conjugated Ly6G (Cat# 560599), PE-conjugated CD11b (Cat# 553311), PE-conjugated CD19 (Cat# 553786), and APC/Cy7-conjugated NK1.1(Cat# 560618) (BD Biosciences, San Diego, CA, USA). PerCP/Cy5.5-conjugated F4/80 (Cat# 157317), APC/Cy7-conjugated Ly6C (Cat# 128025), FITC-conjugated CD14 (Cat# 123308), (Biolegend, San Diego, CA, USA), and PE/Cy7-conjugated CD3 (Cat# 25-0031-82) (eBioscience, San Diego, CA, USA). Cells were blocked with FcR blocker (BD Pharmingen) and then incubated with the indicated antibodies under standard protocols. All samples were analyzed using flow cytometry (FACSVerse system, BD Biosciences) with FlowJo (version 7.6.1) software.
4
Multiparameter Flow Cytometry of Macrophages
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BMDMs were resuspended and stained with PE-Cy7-conjugated anti-CD11c (eBioscience clone N418, 25-0114), FITC-conjugated anti-F4/80 (eBioscience clone BM8, 11-4801), and PE-conjugated CD11b (BD Pharmingen, San Jose, CA, clone M1/70 553311). BD and eBioscience antibodies to CD45 were used as single-stained controls. Data were collected on a BD LSRFortessa flow cytometer running FACSDiva software and analyzed in FlowJo (TreeStar, Ashland, OR). Figures were made in Adobe Creative Suite (San Jose, CA).
5
Cell Surface Marker Quantification
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Expression of cell surface differentiation markers was quantified by flow cytometry. 1 × 106 cells were collected from cultures and centrifuged at 700 rpm for 5 min. Cell pellets were resuspended in 200 μl 37 °C PBS containing 2.5 μl of phycoerythrin (PE)-conjugated CD11b (both from BD Biosciences, San Jose, CA). Following 1 h incubation at 37 °C cell surface expression levels were analyzed by flow cytometry. PE was excited at 488 nm and emission was collected through 505 long-pass dichroic and 530/30 band-pass filters.
6
GFP+ and mCherry+ Cell Sorting
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All antibodies were purchased from BD PharMingen: PE-conjugated CD11b and APC-conjugated Annexin V. Total cells were Fc-blocked and stained with indicated combinations of antibodies for 30 min on ice, then washed three times and resuspended in 1% FBS/PBS. For apoptosis analysis, cells were resuspended with binding buffer and stained with Annexin V and 7-AAD for 15 min at 25 °C in the dark. The flow cytometric data were collected on a BD Calibur flow cytometer and analyzed using FlowJo software or Summit software. For the fluorescence-activated cell sorting (FACS), GFP+ or mCherry+ cells were sorted out by a cell sorter BD FACSAriaTM (BD Biosciences).
7
Flow Cytometric Characterization of mBM-MSCs
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mBM-MSCs were trypsinized with 0.25% trypsin/0.1% EDTA and washed twice with 10.2 g/l PBS (pH=7.2). Cells were subsequently incubated on ice with the following monoclonal antibodies (1:250): FITC-conjugated CD29 (cat. no. 561796; BD Pharminogen; BD Biosciences), FITC-conjugated CD34 (cat. no. 560238; BD Pharminogen; BD Biosciences), FITC-conjugated CD90 (cat. no. 561973; BD Pharminogen; BD Biosciences), phycoerythrin (PE)-conjugated CD44 (cat. no. 553134; BD Pharminogen; BD Biosciences), PE-conjugated CD45 (cat. 553081; BD Pharminogen; BD Biosciences) or PE-conjugated CD11b (cat. no 12-0112-82; eBioscience; Thermo Fisher Scientific, Inc.). PE- and FITC-conjugated IgM and IgG were used as controls. Labeled cells were analyzed by flow cytometry using a BD FACSCaliburä flow cytometer (BD Biosciences) and FlowJo version 10 software (Tree Star, Inc.).
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