Peritoneal exudate cell (PEC) suspensions were blocked with Fc Block (BD Biosciences, San Jose, CA, USA) and subsequently stained with specific Abs including: anti-Ly6G FITC, anti-CD11cPE, anti-CD11b PerCP/CY5.5, Siglec-F PE, anti-c-kit APC, F4/80 APC, anti-CD4APC, anti-CD19 PerCP/Cy5.5, CD3PE (BD Biosciences, San Jose, CA, USA) and anti-CD206PE or anti-CD206 Alexa fluor 488 (Biolegend, USA). Cells were collected 4 hours after microparticle inoculation for analysis of phosphorylation of BTK (Y-551) or SYK (Y-348). Intracellular staining was performed using anti-mouse phospho-BTK/ITK (Y551/Y511) PE (eBiosciences, San Diego, CA, USA), anti-mouse phospho-SYK (Y-348) PE antibody (BD Biosciences, San Jose, CA, USA) and FOXP3/Transcription Factor Staining Buffer Set, which was used as per the manufacturer instructions (eBiosciences, San Diego,CA, USA). For CFSE-labeled cells, anti-CD4 PerCP (BD Biosciences, San Jose, CA, USA), and KJ1–26 PE (BD Biosciences, San Jose, CA, USA) were used to phenotype DO11.10 T cells and cell cycle progression was examined as previously discussed10 (link). For FACS analysis of whole synovial tissues, samples were digested at 37°C for 1 hour with 0.1% collagenase in RPMI supplemented with 10% FBS, 1000U/ml penicillin, 1000U/ml streptomycin, and 2nm L-glutamine. Single cell suspensions were then prepared and blocked and stained as described above.
Anti cd11b percp cy5
Manufactured by BD
Sourced in United States
Anti-CD11b-PerCP-Cy5.5 is a fluorescently-labeled monoclonal antibody that binds to the CD11b cell surface antigen. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd206 pe, by BioLegend (4 mentions)
Anti mhcii fitc, by BD (4 mentions)
Anti cd4 apc, by BD (3 mentions)
Siglecf pe, by BD (3 mentions)
Anti c kit apc, by BD (3 mentions)
F4 80 apc, by BD (3 mentions)
Fc block, by BD (3 mentions)
Anti ly6g fitc, by BD (3 mentions)
Anti cd11c pe, by BD (2 mentions)
Anti b220 apc, by BD (2 mentions)
Anti ly6c pe, by BD (2 mentions)
Anti ly6g apc cy7, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Anti cd8 pe cy7, by BD (2 mentions)
Fortessa x20 flow cytometer, by BD (2 mentions)
Anti cd11c apc, by Thermo Fisher Scientific (2 mentions)
Anti cd45 apc cy7, by BD (2 mentions)
Anti cd86 pe cy7, by BD (2 mentions)
Anti cd86 pe, by BD (2 mentions)
Anti cd3 fitc, by BD (2 mentions)
14 protocols using anti cd11b percp cy5
1
Comprehensive Immune Cell Profiling in Murine Tissues
2
Multicolor Flow Cytometry of Murine Immune Cells
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Splenocytes obtained from control or treated tumor bearing mice were liquid-nitrogen frozen and thawed before tests. Then, for lymphoid cell analysis, they were stained in a one-step test with the following fluorophore-labeled anti-mouse monoclonal antibodies (mAbs): anti-CD4-APC (BD Pharmingen, USA, RM4-5), anti-CD8-PE-Cy7 (BD Pharmingen, USA, 53–6.7), anti-CD49b-PE (BD Pharmingen, USA, DX5) and anti-CD19-FITC (BD Pharmingen, USA, 1D3). Phenotype analysis was carried out using the Becton Dickinson FACSCalibur apparatus with Cell Quest Software. For myeloid cell characteristics, the flow cytometry analysis of MDSC surface phenotype was performed as described previously [16 (link)] using fluorophore-labeled anti-mouse mAbs: anti-CD11b-PerCP-Cy5.5 (BD Pharmingen, USA, M1/70), anti-B220-APC (BD Pharmingen, USA, RA3-6B2), anti-Ly6G-APC-Cy7 (BD Pharmingen, USA, 1A8), anti-Ly6C-PE (BD Pharmingen, USA, AL-21) and anti-MHCII-FITC (BD Pharmingen, USA, 25-9-17). The cells were stained for 45 min at 4°C. The viability of spleen cells was assessed by incubation with DAPI dye. The analysis was carried out using Becton Dickinson FACSFortessa apparatus with FACSDiva software.
3
Lymph Node Cell Phenotyping in Murine Colitis
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Mesenteric lymph node cell suspensions were collected and prepared from Hp1’, and 2′ inoculated Villincre controls and VillinCre-A2BARfl/fl; washed; blocked with Fc Block; and stained with anti-CD4-APC (RM4-5,), anti-pSTAT6-PE (pY641). Phosphorylation of STAT6 at tyrosine 641 was detected by intracellular staining with PE-conjugated anti-phospho-STAT6 using PhosFlow Fix Buffer I and Perm Buffer III reagents. Peritoneal exudate cell (PEC) suspensions were blocked with Fc Block (BD Biosciences, San Jose, CA, USA) and subsequently stained with specific Abs, including anti-Ly6G FITC, anti-CD11b PerCP/CY5.5, Siglec-F PE, anti-c-kit APC, F4/80 APC, BD Biosciences, San Jose, CA, USA) and anti-CD206PE (Biolegend, USA). Cells were acquired on Fortessa X-20 Flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Becton Dickinson & Company, Ashland, OR)
4
Epidermal Cell Isolation and Analysis
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Skin from the footpad was harvested using scalpels. The epidermis and dermis layers were separated after incubation at 37°C for 1 hr in 0.4 mg/ml dispase II (Roche). The epidermis was collected and then cut into small pieces using scalpels and incubated for 30 min with agitation at 37°C in RPMI containing 10% FCS and 0.5 mg/ml DNAse I. Suspensions were filtered using 40 μm cell strainers.
For Langerhans cells analysis, after centrifugation (450 × g, 5 min) cells were stained with anti-CD45 FITC (BioLegend, clone 30-F11), anti-EpCAM APCFire750 (BioLegend, clone G8.8) and anti-CD11b PerCPCy5.5 (BD Biosciences, clone M1/70).
For RNA-seq analysis, after centrifugation (150 × g, 5 min), dead cells were removed using EasySep Dead cell removal kit (StemCell). After this step, viability was around 70% as assessed by flow cytometry. Epidermal cells were composed of 95% keratinocytes (CD45- cells) as assessed by flow cytometry.
For Langerhans cells analysis, after centrifugation (450 × g, 5 min) cells were stained with anti-CD45 FITC (BioLegend, clone 30-F11), anti-EpCAM APCFire750 (BioLegend, clone G8.8) and anti-CD11b PerCPCy5.5 (BD Biosciences, clone M1/70).
For RNA-seq analysis, after centrifugation (150 × g, 5 min), dead cells were removed using EasySep Dead cell removal kit (StemCell). After this step, viability was around 70% as assessed by flow cytometry. Epidermal cells were composed of 95% keratinocytes (CD45- cells) as assessed by flow cytometry.
5
Lymph Node Cell Phenotyping in Murine Colitis
6
Isolation and Characterization of Immune Cells from Mouse Spinal Cord
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The spinal cord, dissected from mice transcardially perfused with PBS, was minced into 1 mm3 pieces in solution containing 1 mg/mL or 0.1 mg/mL collagenase IV (Worthington Biochemical Corporation, USA) plus 0.4 mg/mL DNase I (Roche, Switzerland), and incubated at 37 °C for 15 min. For isolation of immune cells and microglia, cells were resuspended in 37% Percoll (GE Healthcare, USA) and centrifuged at 780 × g for 20 min. After centrifugation, myelin debris was removed and the cell pellet was collected. Cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then stained with combinations of the following antibodies: anti-CD45-PE-Cy7, anti-CD45-APC-Cy7, anti-CD4-PE, anti-CD8a-FITC, anti-CD8a-APC-Cy7, anti-NK1.1-PErCP-Cy5.5, anti-NK1.1-PE, anti-CD11c-PE, anti-CD11b-APC-Cy7, anti-CD11b-PerCP-Cy5.5 (BD Biosciences, USA), anti-CD4-APC, anti-CD3e-PerCP-Cy5.5, anti-CD86-APC, anti-CD25-PE (eBioscience, USA), anti-CD11c-APC, anti-I-A/I-E (MHC class II)-FITC, anti-CD4-PE-Cy7, anti-CD4-APC, anti-CD68-PE, anti-H-2Kb/H-2Db (MHC class I)-PE, anti-CD206-PE, anti-CD178 (FasL)-PE, and Armenian Hamster IgG Isotype control-PE (BioLegend, USA). Flow cytometry was performed by using FACS Aria and FACS Verse flow cytometers (BD Biosciences, USA) and the data was further analyzed using FlowJo Software (FlowJo, LLC, USA).
7
Dendritic Cell Maturation Assay
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Immature BMDC were cocultured with mitomycin C-treated MC38/0, MC38/shN, and MC38/shTGFβ1-1, 2, 3 cells in the presence of GM-CSF (40 ng/ml). After 24 h, dendritic cells were harvested and labeled with monoclonal antibodies conjugated with fluorochromes (BD Biosciences): anti-CD40 PE, anti-CD80 APC, anti-CD86 PE-Cy7, anti-MHC II FITC, anti-CD11b PerCP-Cy5.5, and anti-CD11c BV650. The expression of cell surface markers was analyzed using FACS Calibur with CellQuest software (Becton Dickinson).
8
Flow Cytometric Analysis of Intestinal Macrophages
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Cells were isolated from the jejunum as described above. Cellular debris was removed from lamina propria mononuclear cells using Percoll gradient (Sigma-Aldrich). Antibodies for flow cytometry were purchased from eBioscience (Waltham, MA, USA) unless otherwise stated. Dead cells were excluded from analysis with a viability dye eFluor 780. Macrophages in the lamina propria were identified through staining with anti-major histocompatibility complex (MHC) class II (eFluor 450), anti-CD11b (PerCP/Cy5.5), and anti-F4/80 (BV605; BD Biosciences, San Diego, CA, USA) antibody. We also performed staining with anti-CHI3L1 (PE; Biorbyt, Cambridge, UK) antibody to determine if CHI3L1 is expressed in lamina propria macrophages. Stained cells were detected by flow cytometry using an LSR Fortessa™ X-20 cell analyzer (BD Biosciences), and data were analyzed using FlowJo software (version 10.6.0; Tree Star, Inc., Ashland, OR, USA). The gating strategy is shown in Supplementary Fig. S1 .
9
Multiparametric Flow Cytometry of Tumor Microenvironment
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Spleen and tumor cells obtained from control or treated tumor-bearing mice were thawed, centrifuged, and incubated with monoclonal antibodies conjugated with fluorophores: anti-CD45 V500, anti-CD11b PerCP-Cy5.5, anti-CD11c BV605, anti-CD4 APC, anti-B220 APC, anti-CD49b APC, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHCII FITC, and anti-CD86 PE-Cy7 (all from BD Biosciences). After incubation, cells were suspended in PBS with DAPI dye (Molecular Probes) and analyzed using LSR Fortessa with Diva Software (Becton Dickinson) according to the procedure described by Rossowska and coworkers (27 (link)).
10
Immune Cell Activation and Characterization
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Lipopolysaccharide (LPS) (Escherichia coli, E. coli 055:B5) and PMA [phorbol 12-myristate 13-acetate ≥ 99% (TLC), film or powder] were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ionomycin (Free Base) was purchased from MKbio (Shanghai, China). Recombinant mouse GM-CSF (granulocyte-macrophage colony stimulating factor), IL-4 (interleukin 4) and IL-2 (interleukin 2) were obtained from Peprotech (Rocky Hill, NJ, USA). Cell staining was performed by using the following mAbs from BD Biosciences (Franklin Lakes, NJ, USA): anti-CD45 FITC (30-F11, #553079), anti-CD11b PerCP-Cy5.5 (M1/70, #550993), anti-CD11c APC (HL3, #550261), anti-F4/80 BV421 (T45-2342, #565411), anti-Ly-6G (Gr-1) PE (1A8, #551461), anti-CD80 PE (16-10A1, #553769), anti-MHC II BV421 (M5/114.15.2, #562564), anti-CD86 PE (GL1, #561963), anti-CD40 BV421 (3/23, #562846), anti-CD3 FITC (17A2, #561798), CD4 PerCP-Cy5.5 (RM4-5, #550954), anti-CD8a PerCP-Cy5.5 (53-6.7, #551162), anti-IL-2 PE (JES6-5H4, #554428), anti-TNFα BV421(MP6-XT22, #563387) and anti-IFNγ APC (XMG1.2, 554413). The isotype-matched mAbs for control staining were from BD Biosciences. Cytotoxicity LDH Assay Kit-WST was purchased from Dokindo (Tokyo, Japan).
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