Bone marrow cells were flushed from femurs and tibias, centrifuged, then exposed to geys buffer for 5 min on ice, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with antibodies for 20 min at 4 °C, and washed before sorting CD115+/CD11c-/NK- monocytes using an Influx cell sorter (BD) [68 (link)]. Monocytes were cultured in complete RPMI medium (Thermo Fisher Scientific) and exposed to 100 ng/mL CSF1, 50 µM Q-VD-OPh and 50 µM Ac-YVAD-cmk. After five days, macrophages were studied by flow cytometry using AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BUV737-CD43 (#564398, BD). For the sorting of monocytes, the antibodies used were the following: APC-CD11b (#17-0112-83, Thermo Fisher Scientific), PE/Cy7-CD11c (#561022, BD), FITC-NK (#553164, BD), PerCP/Cy5.5-Ly6C (#560525, BD), AlexaFluor700-Ly6G (#561236, BD), biotin-CD115 (#135507, Biolegend), PE-Streptavidin (#554061, BD). The data were analyzed with FlowJo software v. 10.0.00003.
Cd54 apc
Manufactured by BioLegend
CD54-APC is a fluorescent-labeled antibody that binds to the cell surface protein CD54, also known as ICAM-1. It can be used for the identification and analysis of cells expressing CD54 in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd11c pe cy7, by BD (2 mentions)
Cd71 pe, by BioLegend (2 mentions)
Siglecf pe cf594, by BD (2 mentions)
Cd11b bv510, by BioLegend (2 mentions)
Gr1 apc cy7, by BioLegend (2 mentions)
Cd206 pe cy7, by BioLegend (2 mentions)
Murine trustain fcx, by BioLegend (2 mentions)
Alexafluor700 f4 80, by Bio-Rad (2 mentions)
Percp cy5.5 cd11c, by BD (2 mentions)
Pb cd40, by BioLegend (2 mentions)
Bv711 cd64, by BioLegend (2 mentions)
Bv605 ia ie, by BD (2 mentions)
Buv737 cd43, by BD (2 mentions)
Complete rpmi medium, by Thermo Fisher Scientific (1 mentions)
Cd11b apc, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe, by BD (1 mentions)
Ly6g alexa fluor 700, by BD (1 mentions)
Influx cell sorter, by BD (1 mentions)
Percp cy5.5 ly6c, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
5 protocols using cd54 apc
1
Monocyte-derived Macrophage Phenotyping
2
Interstitial Macrophages Identification
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Right lungs were digested with the Lung Dissociation kit (Miltenyi Biotec), filtered, erythrocytes were removed using ACK and nucleated cells were collected. Cells were washed with ice-cold PBS, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with the antibodies for 20 min at 4 °C, washed, then fluorescence was measured with a BD LSRFortessa X-20. Antibodies used were the following: AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), FITC-CD45 (#103108, Biolegend), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BV650-CD24 (#563545, BD), BUV737-CD43 (#564398, BD). The data were analyzed with FlowJo software v. 10.0.00003. Interstitial macrophages were selected according to their larger size (FSC) and granularity (SSC), on CD45 expression to select leukocytes and GR1-positive cells will be excluded to remove neutrophils, they are equally CD11b high, SiglecF negative, IA-IE positive and CD24 negative.
3
Comprehensive Immunophenotyping of T Cell Subsets
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Blood was collected and processed within 4 hours maximum. An ammonium chloride-based lysing reagent (BD Pharm Lyse, BD Biosciences) was used for erythrocyte deletion of 1ml of blood. After washing, cells were suspended in PBS and stained with Aqua Dye (Invitrogen) for cell viability. Cells were washed again, suspended in staining buffer and divided in four tubes. The four different panels assessed contained some common and some specific antibodies. Common antibodies were: CD3-eFluor 605, CD4-Alexa700 (eBioscience, San Diego, CA), CCR7-Horizon PE-CF594, CD38-Brillant Violet 421, HLA-DR-PerCP-Cy5.5 and CD11c-PE-Cy7 (BD Biosciences). Specific for each panel were: 1) CCR2-PE, CCR5-APC-Cy7, CXCR6-APC (R&D Systems Inc.) and CXCR3-FITC (BioLegend); 2) CD49d (α4)-FITC, β7-APC, CCR9-PE (BD Biosciences) and CD29 (β1)–APC-Cy7 (BioLegend); 3) CD103-FITC, CD54-APC, CD49a (α1)-PE and CD29–APC-Cy7 (BioLegend); 4) CD18-APC, CLA-FITC (BD Biosciences) and CCR10-PE (BioLegend). Cells were acquired using a BD LSRFortessa SORP flow cytometer (Flow Cytometry Platform, IGTP) and analyzed with FlowJo 9.3.2 software (TreeStar). Gates were drawn based on fluorescence minus one-controls and isotypes, and CD3+ CD4- phenotype was considered CD8+ T cells.
4
Flow Cytometric Analysis of CD11b Knockdown
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Flow cytometric analysis was performed on a Fortessa or FACSCalibur FACS machine (BD). Standard staining was performed. In brief, cells were washed three times with FACS buffer and blocked using Fc-block CD16/32 antibody (BioLegend) for 15 min, followed by incubation with fluorescently labeled antibody for 30 min. Analysis was performed using FlowJo software (version 9.2; Tree Star). PBMCs were isolated by Ficoll centrifugation. The antibodies used were: CD11b-PE antibody (BioLegend), CD162–Alexa Fluor 647 (BD), CD62L-FITC and CD18-PE (eBioscience), and CD54-APC, CD14-Pe/Cy7, CD49d-FITC, CD11a-PE, CD11b–eFluor 450 (all BioLegend). Sterile FACS to maintain CD11b knockdown in CD11bkd BV2 cell lines was performed after staining of CR3kd and control cells with CD11b-PE antibody, as described in the previous paragraph. Cells were sorted on a FACSAriaIII cell sorter (BD) for CD11blow-expressing cells, defined by CD11b expression of <80% of control cells. Then, cells were cultured as described in the Cell lines section.
5
Isolation and Characterization of Mouse Muscle Stem Cells
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