Cells harvested from the spleen were used for single color and multi-color staining controls. Control and tumor samples were resuspended at a concentration of 106 cells/100 uL in FWB in round bottom polystyrene tubes. Cells were stained with the following antibody fluorophore conjugates: CD45-PerCP/Cy5.5, CD11c-Pacific Blue, CD11b-AF700, MHCII-APC/Cy7, Ly6c-BV510, Ly6g-APC, CD38-FITC (Biolegend, San Diego, CA), F480-PE/Cy7 (Tonbo Bioscences, San Diego, CA), CD206-PE (R&D Systems, Minneapolis, MN) and propidium iodide (PI, Enzo Life Sciences, Farmingdale, NY). All antibody staining took place for 30 min at 4 ° C in the dark. For all experiments, cells were acquired on the BD LSRII Fortessa flow cytometer. Compensation and sequential gating was performed with FlowJo software (FlowJo LLC, Ashland, OR). Populations of myeloid cells that were identified included macrophages, dendritic cells, granulocytic myeloid derived suppressor cells (G-MDSC), monocytic myeloid derived suppressor cells (M-MDSC) and M0, M1 and M2 macrophage phenotypes (Supplementary Fig. S1 ).
Cd11b af700
Manufactured by BioLegend
CD11b-AF700 is a fluorescently-labeled antibody that binds to the CD11b protein, a cell surface marker expressed on various immune cells, including monocytes, macrophages, and granulocytes. This product is suitable for flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ly6g pe, by BioLegend (3 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Ifn γ percp cy5, by BD (2 mentions)
Cd4 pe f594, by BD (2 mentions)
Il 17a rat pe cy7, by BioLegend (2 mentions)
Il 17a alexa fluor 488, by BD (2 mentions)
Ifn γ allophycocyanin cy7, by BioLegend (2 mentions)
Il 10 allophycocyanin, by BioLegend (2 mentions)
Il 6 allophycocyanin, by BioLegend (2 mentions)
T bet pe, by BioLegend (2 mentions)
Lsrii flow cytometer, by Tree Star (2 mentions)
Zombie aqua, by BioLegend (2 mentions)
Cd45 apc cy7, by BioLegend (2 mentions)
Ly6g apc, by BioLegend (1 mentions)
Lsrii fortessa flow cytometer, by BD (1 mentions)
Cd38 fitc, by BioLegend (1 mentions)
Propidium iodide, by Enzo Life Sciences (1 mentions)
Cd45 percp cy5, by BioLegend (1 mentions)
Ly6c bv510, by BioLegend (1 mentions)
Mhcii apc cy7, by BioLegend (1 mentions)
6 protocols using cd11b af700
1
Multicolor Flow Cytometry of Myeloid Cells
2
Comprehensive Cytokine and Lymphocyte Analysis
3
Quantifying Dendritic Cells and Th17 Cells
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Single-cell suspensions were initially blocked with an Fc receptor blocker, anti-mouse CD16/32 (Thermo Fisher Scientific, Waltham, MA), on ice for 10 minutes, then stained with fluorochrome-conjugated antibodies for 15 minutes in the dark in accordance with previously established protocols (18 (link), 19 (link)). All flow cytometry analyses were conducted exclusively on the live cell populations, which was achieved by excluding dead cells through staining with Zombie Aqua (BioLegend, San Diego, CA). To quantify type 1 conventional dendritic cells (cDC1) and T helper-17 (Th17) cells, the following anti-mouse primary antibodies were utilized: CD45-APC/Cy7, CD11c-BV421, CD11b-AF700, MHCII-FITC, Ly6C-APC-Cy7, Ly6G-PE, CD3-FITC, CD4-APC, FOXP3-BV421, and RORγT-PE (BioLegend). Activated cDC1 cells were identified as CD45+ Ly6G- MHCII+ CD11c+ CD11b- CD86+. Th17 cells were identified as CD45+ CD3+ CD4+ RORγT+. Analysis was carried out using a BD FACSAria Fusion flow cytometer (BD Biosciences, San Jose, CA), and flow cytometry data were subsequently analyzed using the FlowJo software (FlowJo, Ashland, OR).
4
Murine Lung Immune Cell Profiling
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On day 7, the lungs were isolated from mice, grounded, and subsequently passed through a 70μm cell strainer. Ammoniumchloride-potassium lysing buffer was used for erythrocyte lysis. Multiparameter assessments were performed using BD FACSAria II (BD Biosciences) and data was analyzed with Flowjo software (version 10.6). After staining Zombie Aqua™ (Biolegend) to exclude dead cells, single-cell suspensions were incubated with the fluorochrome-conjugated antibodies in PBS containing 2% fetal bovine serum and then washed twice before detection. Antibodies specific to mouse used included CD16/32 (BD, 553,141), CD45-APC-Cy7 (Biolegend, 103,116), CD11b-AF700 (Biolegend, 101,222), Ly6G-PE (Biolegend, 127,608), CD62L-BV650 (Biolegend, 104,453), CD3-BV711 (Biolegend, 100,241), CD19-BV711 (Biolegend, 115,555), NK1.1-Pacific Blue (Biolegend, 109,816).
5
Comprehensive Cytokine and Lymphocyte Analysis
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Lymphocytes were all stained with the anti-mouse Abs CD4 PE-F594 (BD Biosciences, San Jose, CA), CD4 FITC (BioLegend, San Diego, CA), CD11b AF-700 (BioLegend), and GL3 (γδ TCR). All cytokine staining was performed using the BD Foxp3 intranuclear transcription factor staining kit for Foxp3 PE (eBioscience), T-bet PE (BioLegend), IL-17a Rat PE-Cy7 (BioLegend), IL-17a Alexa Fluor 488 (BD Biosciences), IFN-γ PerCP-Cy5.5 (BD Biosciences), IFN-γ allophycocyanin-Cy7 (BioLegend), IL-10 allophycocyanin (BioLegend), and IL-6 allophycocyanin (BioLegend) following the manufacturer’s instructions. For phenotypic analysis, flow cytometry was performed using a BD Biosciences LSR II flow cytometer, and analysis was performed using FlowJo (Tree Star, Ashland, OR).
6
Isolation and Analysis of Tumor-Associated Immune Cells
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The carcinogen 4-nitroquinoline-1-oxide (4NQO) was purchased from Sigma–Aldrich and diluted in sterile water to a final concentration of 50 μg/mL or 100 μg/mL. The in vivo anti-mouse Ly6G/Ly6C (Gr1) neutralizing antibody was obtained from BioXcell. Palmitic acid (PA), oleic acid (OA), and oil red O were provided by Sigma–Aldrich. DNase I was acquired from Biofroxx. FcR-Blocking Reagent (anti-mouse CD16/32), Zombie GreenTM Fixable Viability Kit and the following fluorescently labeled antibodies were from BioLegend: anti-mouse mAbs CD45-BV605, Ly6G/Ly6C (Gr1)-BV510, Gr1-AF594, Ly6C-BV421, Ly6G-PE, CD3-APC/Cy7, CD4-PE/Cy7, CD8a- PerCP/Cy5.5, CCR1 (CD191)-APC, CD11b-APC, and CD11b-AF700. RBC lysis buffer, IC fixation buffer, permeabilization buffer (10×), and anti-mouse mAbs Arg1-APC and iNOS-PE were obtained from eBioscience. The CellTrace™ Violet Cell Proliferation Kit, BODIPYTM 493/503, and immunostaining antibody against CCR1 were from Life Technologies. Rabbit anti-mouse Ki67 antibody was purchased from Arigo Biolaboratories. Primary anti-mouse/human CD11b, GAPDH, anti-human CD33, and anti-mouse MIP1γ (CCL9) antibodies were purchased from Abcam. Immunostaining antibody against Ly6G was acquired from Servicebio. CCL9 protein was from NovoProtein. Secondary antibodies coupled to fluorescent Alexa probes were purchased from Beijing Emarbio Science Technology.
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