On day 7 after laser-induced CNV, mice were transcardially perfused with iced-cold PBS. After eyes were quickly removed, the retina and RPE-choroid-sclera complexes were cut into small pieces. The tissue was further mechanically dissociated by trituration and the suspension was applied to 30 um cell strainer. The single cells were pre-incubated with Fc-block followed by stained with FITC-conjugated anti-CD11b (#101,206, BioLegend), APC-conjugated anti-CD80 antibodies (#104,714, BioLegend), PE-conjugated anti-CD80 (#104,708), PE-conjugated anti-CD206 (#141,706, BioLegend) and/or APC-conjugated anti-CD206. Stained cells were processed using LSR-II cytometer (BD Biosciences), and the data were analysed using FlowJo Software (version 7.6.2).
Fitc conjugated anti cd11b
Manufactured by BioLegend
Sourced in United States
FITC-conjugated anti-CD11b is a monoclonal antibody that binds to the CD11b cell surface antigen. CD11b is expressed on the surface of various immune cells, including monocytes, macrophages, and neutrophils. The FITC fluorescent label allows for the detection and analysis of CD11b-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Fitc conjugated anti gr 1, by BioLegend (2 mentions)
Pe conjugated anti cd80, by BioLegend (1 mentions)
Pe conjugated anti cd206, by BioLegend (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Fitc conjugated anti ifn γ, by BioLegend (1 mentions)
Pe conjugated anti il 13, by Thermo Fisher Scientific (1 mentions)
Percp cy5.5 conjugated anti il 17a, by BioLegend (1 mentions)
Pe conjugated anti b220, by Thermo Fisher Scientific (1 mentions)
Percp cy5.5 conjugated anti cd8, by BioLegend (1 mentions)
Fitc conjugated anti cd44, by BioLegend (1 mentions)
Pe conjugated anti cd62l, by BioLegend (1 mentions)
Apc conjugated anti cd45, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Apc conjugated anti cd4, by BioLegend (1 mentions)
Anti il 1β, by Thermo Fisher Scientific (1 mentions)
Cyan adp, by Beckman Coulter (1 mentions)
Apc conjugated anti tnfα, by Thermo Fisher Scientific (1 mentions)
Fixation and permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
12 protocols using fitc conjugated anti cd11b
1
Characterization of Immune Cells in Laser-Induced CNV
2
Multiparameter Flow Cytometry Analysis of Lymphocyte Subsets
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Cells were harvested and stimulated for 4 h with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend). For intracellular staining, cells were fixed and permeabilized (eBioscience) and then stained with FITC-conjugated anti–IFN-γ (505806, BioLegend), PE-conjugated anti–IL-13 (12-7133-81, eBioscience), PerCP/Cy5.5-conjugated anti–IL-17A (506919, BioLegend), and APC-conjugated anti-FOXP3 (17-5773-80, eBioscience) antibodies. To analyze lymphocyte development in Ptenfl/flIl17acre mice, cells isolated from the thymus, pLNs, and spleen were stained with FITC-conjugated anti-CD3ε (11-0031-81, eBioscience), PE-conjugated anti-B220 (12-0451-82, eBioscience), PerCP/Cy5.5-conjugated anti-CD8 (126609, BioLegend), APC-conjugated anti-CD4 (100411, BioLegend), FITC-conjugated anti-CD44 (103006, BioLegend), PE-conjugated anti-CD62L (104407, BioLegend), and PerCP/Cy5.5-conjugated anti-TCR γ/δ (118117, BioLegend) antibodies. For other surface antigen staining, cells were stained with APC-conjugated anti–IL-6Rα (115811, BioLegend), APC-conjugated anti-CD45 (103111, BioLegend), FITC-conjugated anti–GR-1 (108405, BioLegend), and FITC-conjugated anti-CD11b (101205, BioLegend) antibodies. Stained cells were then analyzed using a FACSCalibur flow cytometer (BD Bioscience), and data were analyzed with FlowJo software.
3
Cytokine Expression Profiling in Mouse N-9 Cells
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To measure cytokine expression, mouse N-9 cells were cultured with 5 µg/ml brefeldin A (eBioscience), which prevents protein transportation and leads to cytokine accumulation intracellularly. Cells were surface-stained with FITC-conjugated anti-CD11b (BioLegend, San Diego, CA) followed by intracellular staining of APC-conjugated anti-TNFα (eBioscience) and anti-IL-1β (eBioscience) with fixation and permeabilization buffer (eBioscience). The stained samples were analyzed on a Beckman Coulter CyAn ADP and data were analyzed with FlowJo software.
4
Characterizing Myeloid Cell Differentiation
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Kasumi-1 cells were plated in 6-well-plates (5x105 cells/well) and treated for 6 days with the indicated concentrations of DAC and/or BZ. DAC and/or BZ were administrated fresh every 24 h, along with fresh medium. Untreated cells were plated at a density of 3x105 cells/well. After washing and blocking with FcBlocker (cat. no. 422302; BioLegend, Inc.) for 10 min at room temperature, cells were stained in three 4-color tubes with FITC-conjugated anti-CD11b (cat. no. 301329; BioLegend, Inc.), anti-CD14 (cat. no. CYT-14F6; Cytognos), anti-CD15 (cat. no. CYT-15F4; Cytognos), phycoerythrin (PE)-Cyanine 5-conjugated anti-CD16 (cat. no. 302010; BioLegend, Inc.) and anti-HLA-DR (cat. no. 307608; BioLegend, Inc.), allophycocyanin-conjugated anti-CD45 (cat. no. CYT-45AP5; Cytognos) and PE-conjugated anti-CD13 (cat. no. 301704; BioLegend, Inc.), anti-CD117 (cat. no. 313204; BioLegend, Inc.) and anti-CD193 (cat. no. 310706; BioLegend, Inc.). The analysis was performed on a CyFlow ML (Partec).
5
Immune Cell Profiling by Flow Cytometry
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The immune cell populations were subjected to surface staining for flow cytometry analysis. The used antibodies are as follows: PE‐conjugated anti‐CD3ε (Biolegend), PerCP‐conjugated anti‐CD4 (BD Pharmingen), PerCP‐conjugated anti‐CD8α (BD Pharmingen), FITC‐conjugated anti‐NK1.1 (Biolegend), FITC‐conjugated anti‐CD11b (Biolegend), PE‐conjugated anti‐CD103 (Biolegend), APC‐conjugated anti‐CD11c (BD Pharmingen), PerCP‐conjugated anti‐F4/80 (Biolegend), or FITC‐conjugated anti‐Gr‐1 (Biolegend).
For analyzing cytokine secretion, splenocytes were inoculated into a 12‐well plate at 4 × 106/well and incubated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 3 days, then added 500 ng/mL Ionomycin (Sigma‐Aldrich) and 50 ng/mL PMA (Sigma‐Aldrich) and 5 ng/mL Brefeldin A (BFA, eBioscience) to incubate for 5 hours. Then cells were collected and operated extracellular staining with anti‐mouse PerCP‐conjugated anti‐CD8α and intracellular staining with following anti‐mouse antibodies: APC‐conjugated anti‐IFN‐γ (BD Pharmingen), PE‐conjugated anti‐IL‐2 (BD Pharmingen), FITC‐conjugated anti‐TNF‐α (BD Pharmingen). The data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software were analyzed by FlowJo software (Tree Star Inc).
For analyzing cytokine secretion, splenocytes were inoculated into a 12‐well plate at 4 × 106/well and incubated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 3 days, then added 500 ng/mL Ionomycin (Sigma‐Aldrich) and 50 ng/mL PMA (Sigma‐Aldrich) and 5 ng/mL Brefeldin A (BFA, eBioscience) to incubate for 5 hours. Then cells were collected and operated extracellular staining with anti‐mouse PerCP‐conjugated anti‐CD8α and intracellular staining with following anti‐mouse antibodies: APC‐conjugated anti‐IFN‐γ (BD Pharmingen), PE‐conjugated anti‐IL‐2 (BD Pharmingen), FITC‐conjugated anti‐TNF‐α (BD Pharmingen). The data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software were analyzed by FlowJo software (Tree Star Inc).
6
Validating Bone Marrow Macrophage Differentiation
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For evaluate whether bone marrow-derived macrophages were correctly differentiated, 9 d-cultured cells were stained with anti-macrophage antibodies; PE conjugated anti-F4/80 (BD Bioscience, San Jose, CA) and FITC conjugated anti-CD11b (BioLegend, San Diego, CA). Almost all of the BM-derived cells after 9 days of differentiation were F4/80+CD11b+. (Fig 1A and 1B ). A FACScan flow cytometer (BD Biosciences, San Jose, CA) was used to acquire 10,000 events for each sample.
7
Evaluating Cell Surface Markers by Flow Cytometry
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The cell surface markers of differentiation were evaluated by flow cytometry as described in our previous study [16 (link)]. AML cell lines or human primary AML cells were cultured in 6-well plates at a density of 1 × 106 cells per well and treated with different compounds for 72 h. Subsequently, treated cells were resuspended in phosphate-buffered saline containing 1 mM EDTA and 2% FBS, and incubated with FITC-conjugated anti-CD11b or PE-conjugated anti-CD14 (BioLegend) for 30 min in the dark at room temperature. Flow cytometer data were collected by BD FACS Caliber (BD Biosciences, NJ, USA) and analyzed through FlowJo (BD Biosciences, NJ, USA).
8
Quantifying Peritoneal Immune Cells
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Cells collected from peritoneal lavage fluids was washed twice by cold PBS, and then stained with FITC-conjugated anti-CD11b (Biolegend, San Diego, CA, USA) and PE/Cy7-conjugated anti-Ly6G (Biolegend, San Diego, CA, USA) antibodies diluted in PBS with 2% FBS for 30 min at 4°C. The cells were washed two times with PBS/2% FBS. Finally, CD11b+ PECs and CD11b+ Ly6G+ neutrophils were analyzed by flow cytometer (CytoFLEX, Beckman Coulter, USA).
9
Flow Cytometric Immune Cell Profiling
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Isolated liver leukocytes or PBMCs were blocked with 5 μg/mL anti-mouse CD16/CD32 antibodies (BD Bioscience) and stained with fluorochrome-conjugated antibodies for 30 min at 4 °C in the dark. The cells were washed twice with PBS containing 3% FBS and fixed in 0.5 mL of Fluorofix buffer (BioLegend) for 30 min. The fixed cells were washed with PBS containing 3% FBS once and then analyzed on a FACSFortessa instrument (BD Bioscience). The fluorochrome-conjugated antibodies used in this study included FITC-conjugated anti-CD3, FITC-conjugated anti-CD11b, phycoerythrin-conjugated anti-CD45, APC-conjugated anti-CD8, APC-conjugated anti-CD115, APC/Cy7-conjugated anti-CD45, APC/Cy7-conjugated anti-Ly6G, BV605-conjugated anti-CD4, BV605-conjugated anti-Ly6G, BV421-conjugated anti-Ly6C, PerCP/Cy5.5-conjugated anti-CD19, PerCP/Cy5.5-conjugated anti-F4/80 (all purchased from BioLegend) and BV421-conjugated anti-CD11a (BD Bioscience). The compensation settings of the flow cytometer were determined with single-stained Ultracomp eBeads (Thermo Fisher Scientific). The frequency of each cell subpopulation obtained by flow cytometric analysis was further multiplied by the total isolated cell count to obtain the cell number for each subpopulation.
10
Analysis of Circulating GFP+ and CD105+CD90+CD45-CD11b- Cells
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For the analysis or sorting of GFP+CD45− or CD105+CD90+CD45−CD11b− cells in peripheral circulation, blood samples were collected from mice by intracardiac puncture. After the process of red blood cell lysis with commercial ammonium‐chloride‐potassium lysis buffer (Quality Biological, Gaithersburg, MD, http://www.qualitybiological.com ), cells were washed and counted. We incubated equal numbers of cells for 45 minutes at 4°C with fluorescein isothiocyanate (FITC)‐conjugated anti‐GFP (PA1‐46326; Thermo Fisher Scientific, Waltham, MA, https://www.thermofisher.com ) and peridinin chlorophyll protein (perCP)‐conjugated anti‐CD45 (103132; BioLegend, San Diego, CA, http://www.biolegend.com ). We also incubated cells with phycoerythrin (PE)‐conjugated anti‐Sca1 (108108; BioLegend), allophycocyanin‐conjugated anti‐CD90 (140311; BioLegend), perCP‐conjugated anti‐CD45 (103132, BioLegend), and FITC‐conjugated anti‐CD11b (101208, BioLegend) antibodies. Probes were analyzed using Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, http://www.bd.com ) and CellQuest software.
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