Isolated brain tumour tissue was digested in cysteine-activated papain (Worthington, 50 U ml−1 in HBSS, pH 7) with 300 U ml−1 DNAse for 5 min at 37 °C, triturated, and strained through a 70 µm strainer. Cell suspension was resuspended in HBSS and subsequently underlayed with 35 and 70% Percoll (GE Healthcare) and centrifuged for 15 min at 800 × g. Interphase between layers was collected and washed using PBS with 1% bovine serum albumin and 0.1% NaN3. After Fc receptor blocking (TruStain FcX™ (Clone 93) #101320, BioLegend), cells were incubated with fluorochrome-conjugated primary antibodies at 1:100 dilution. Antibodies for flow cytometry (V450-Ly6G, #560603; PE-Cy7-Ly6C #560593; PerCP-Cy™5.5-CD11b #550993; and APC-CD45 #559864) were purchased from BD Biosciences. The gating strategy is depicted in Supplementary Fig. 1 : we selected CD11bhiCD45+ population and separated Ly6G+ neutrophils and Ly6C+ newly infiltrated monocytes. The CD45lowLy6Clow population is microglial cells and the CD45hiLy6Clow population is cells of myeloid origin, differentiated TAMs. Analysis was performed using CytoFLEX platform (Beckman Coulter) with CytExpert software. Analysis of data was performed using FlowJo software. Fluorescence-activated cell sorting was performed using Sony SH800 cell sorter to collect GFP+ and GFP− populations from NeuroD2:SmoA1;Atoh1:GFP tumours.
Percp cy5.5 cd11b
Manufactured by BD
Sourced in United States
PerCP-Cy5.5-CD11b is a fluorescent-labeled antibody that binds to the CD11b cell surface antigen. It can be used to identify and quantify cells expressing CD11b, such as monocytes and granulocytes, in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
F4 80 pe, by BD (3 mentions)
Trustain fcx clone 93, by BioLegend (1 mentions)
Ly6g v450, by BD (1 mentions)
Ly6c pe cy7, by BD (1 mentions)
Cd45 apc, by BD (1 mentions)
Cytoflex platform, by Beckman Coulter (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Sh800 cell sorter, by Sony (1 mentions)
Cd3 fitc, by BD (1 mentions)
Percp cy5.5 mhcii, by BD (1 mentions)
Cd11b apc, by BD (1 mentions)
Percp cy5.5 cd4, by BD (1 mentions)
Cell stimulation cocktail, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Il 17a pe, by BD (1 mentions)
Cd11c apc, by BD (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd45 fitc, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Ifn γ apc, by BD (1 mentions)
10 protocols using percp cy5.5 cd11b
1
Isolation and Analysis of Brain Tumor Immune Cells
2
Multiparameter Flow Cytometry Analysis
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Anti-mouse antibodies including fluorescein isothiocyanate (FITC)–CD45, PerCP-Cy5.5-MHC-II, PerCP-Cy5.5-CD11b, APC-CD11c, APC-CD11b, R-phycoerythrin (PE)-CD11c, PE-MHC-II, PE-Cyanine7-MHC-II, PE-CD80, BV421-CD86, BV421-IL-10, PE-IL-17A, APC-IFN-γ, FITC-CD3, PerCP-Cy5.5-CD4, and PE-CX3CR1 were obtained from BD Biosciences or BioLegend. CLP and MLN lymphocytes were collected and washed. For surface staining, cells were washed and stained with fluorescent conjugated antibodies for 30 min at 4°C. For intracellular cytokine staining, cells were cultured for 5 hours at 37°C with cell stimulation cocktail (BioLegend). Cells were collected for surface staining and then intracellular staining using the BD Cytofix/Cytoperm Kit (BD Biosciences). After washing with phosphate-buffered saline, the cells were run on a BD LSRFortessa (BD Immunocytometry Systems), and data were analyzed with FlowJo (Tree Star).
3
Multiparametric Leukocyte Phenotyping
4
Isolation and Characterization of Mouse Intestinal Lamina Propria Cells
5
Flow Cytometry Immunophenotyping and Sorting
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In total, 100 μL cell suspension (10
7cells/mL) were collected by centrifugation, followed by staining with 5 μL percp-cy5.5 CD11b, Pacific Blue ly6 g, APC-cy7 ly6c, or APC CD11c antibody (BD Biosciences). The mixture was incubated for 15 to 30 minutes on ice in the dark. Cells were washed twice and collected by centrifugation. The cells were then resuspended in binding buffer and stained for 15 minutes in the dark with annexin V and PI (Annexin V-FITC Apoptosis Detection Kit; BD Biosciences). LSRFortessa flow cytometer (BD Biosciences) was used to analyze the cells. Data were analyzed by using FlowJo-V10 software (Tree Star, Ashland, Oregon, Unite States). Cells were stained as described above and sorted on BD FACS Aria (BD Biosciences).
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In total, 100 μL cell suspension (10
7cells/mL) were collected by centrifugation, followed by staining with 5 μL percp-cy5.5 CD11b, Pacific Blue ly6 g, APC-cy7 ly6c, or APC CD11c antibody (BD Biosciences). The mixture was incubated for 15 to 30 minutes on ice in the dark. Cells were washed twice and collected by centrifugation. The cells were then resuspended in binding buffer and stained for 15 minutes in the dark with annexin V and PI (Annexin V-FITC Apoptosis Detection Kit; BD Biosciences). LSRFortessa flow cytometer (BD Biosciences) was used to analyze the cells. Data were analyzed by using FlowJo-V10 software (Tree Star, Ashland, Oregon, Unite States). Cells were stained as described above and sorted on BD FACS Aria (BD Biosciences).
6
Cell Surface Protein Expression in MSCs
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To analyze the expression of specific cell surface proteins on AD-MSCs and BM-MSCs, flow cytometric analysis was performed (n=5 AD-MSCs and n=5 BM-MSCs). The cells from the third passage were trypsinized into single-cell suspensions and labeled with the following anti-mouse antibodies conjugated to fluorochromes: PerCP-Cy5.5-CD11b (0.25 μg/100 μl; BD Biosciences, Bedford, MA, USA), PE-CD14 (0.25 μg/100 μl; BioLegend, CA, USA), PE-CD34 (0.25 μg/100 μl; BioLegend), PE-CD44 (10 μl/100 μl; BioLegend), APC-CD45 (0.25 μg/100 μl; BioLegend), PE-CD49d (0.25 μg/100 μl; BioLegend), PE-CD73 (0.25 μg/100 μl; BioLegend), PE-CD90.2 (0.25 μg/100 μl; BioLegend), PE-CD105 (0.25 μg/100 μl; Abcam, Cambridge, UK), PE-CD106 (0.25 μg/100 μl; BioLegend), APC-CD133 (0.25 μg/100 μl; BioLegend) and PE-Sca-1 (0.5 μg/100 μl; BioLegend). Cells were incubated on ice for 45 minutes with each antibody. Corresponding mouse isotype antibodies were used as controls (1:5; Santa Cruz Biotechnology, TX, USA). The cells were analyzed using the fluorescence-activated cell sorting instrument (FACSCanto II; BD Biosciences) according to the manufacture’s protocol. The percentage of expressed antigen was calculated for 10,000 gated-cell events and the data processed (FACSDiva software; BD Biosciences).
7
Characterization of MICL Expression in Clec12A Knockout Mice
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MICL knockout mice (Clec12A−/−) on a C57BL/6 background were produced by Taconic Artemis, USA (see online supplementary figure S1A). Clec12A−/− and wild-type (wt) mice were bred and maintained at the Medical Research Facility, University of Aberdeen. Mice were separately housed in groups and provided freely with food and water. All experimentation conformed to the terms and conditions of UK Home Office licences for research on animals (PPL 60/4007) and the University of Aberdeen ethical review committee.
Characterisation of MICL expression in 8–12-week-old wt and Clec12A−/− mice was performed by flow cytometry on cells isolated from the peripheral blood, bone marrow, peritoneal cavity, spleen and lungs, as previously described.14 (link) Antibodies used in these experiments included biotin anti-MICL monoclonal antibody 3097 (link) and isotype control, as well as biotin-Gr-1, FITC-7/4, PE-F480, biotin-F480, PerCpCy5.5-CD11b, PE-CCR3, biotin-NK1.1, PE-CD49, PerCpCy5.5-B220, PE-CD19, PerCpCy5.5-CD3, biotin-CD4, FITC-CD8, biotin-CD11c, PerCpCy5.5-Gr-1, APC-Ly6G, PE-CD11b, PE-Ly6G, PECy7-CD11b, Alexa Fluor 488 anti-STAT5, APC-streptavidin (all from BD Biosciences), and Alexa Fluor 700-F4/80 (BioLegend). Flow cytometry was undertaken using a BD LSRFortessa cell analyser and data analysed using FlowJo.
Characterisation of MICL expression in 8–12-week-old wt and Clec12A−/− mice was performed by flow cytometry on cells isolated from the peripheral blood, bone marrow, peritoneal cavity, spleen and lungs, as previously described.14 (link) Antibodies used in these experiments included biotin anti-MICL monoclonal antibody 3097 (link) and isotype control, as well as biotin-Gr-1, FITC-7/4, PE-F480, biotin-F480, PerCpCy5.5-CD11b, PE-CCR3, biotin-NK1.1, PE-CD49, PerCpCy5.5-B220, PE-CD19, PerCpCy5.5-CD3, biotin-CD4, FITC-CD8, biotin-CD11c, PerCpCy5.5-Gr-1, APC-Ly6G, PE-CD11b, PE-Ly6G, PECy7-CD11b, Alexa Fluor 488 anti-STAT5, APC-streptavidin (all from BD Biosciences), and Alexa Fluor 700-F4/80 (BioLegend). Flow cytometry was undertaken using a BD LSRFortessa cell analyser and data analysed using FlowJo.
8
Immunophenotyping of Tumor-Infiltrating Cells
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The mice were sacrificed before cell line injection, one and four weeks after injection. At this time, blood, lymph nodes in the neck and inguinal area, and spleens were harvested. The lymph nodes and spleens were mechanically dissociated to make single-cell suspensions. The blood was centrifuged and the serum extracted for another study. After red blood cell (RBC) lysis, the precipitate was suspended in flow cytometry buffer. Anti-mouse FITC CD4, PreCP-Cy5.5 CD8, APC CD40, PE CD40L, PerCP-Cy5.5 CD11b, and FITC CD19 (all from BD Bioscience) were used for flow cytometry.
9
Microglial Identification and Isolation
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Following Percoll enrichment, samples were stained for surface antigens, as previously described (Horchar and Wohleb, 2019 (link)). In brief, Fc receptors were blocked with anti-CD16/CD32 antibody (BioLegend, San Diego, CA, U.S.A., #553141). Cells were washed and then incubated with conjugated antibodies (FITC-CX3CR1, #149020 Biolegend; PE-CD115, #565249 BD Biosciences; PE-CF594-CD45, #562420 BD Biosciences; PerCp-Cy5.5-CD11b, #550993 BD Biosciences) for 1 h at 4οC. Cells were washed and then re-suspended in FACS buffer for analysis. Non-specific binding was assessed using isotype-matched antibodies. Antigen expression was determined using a BioRad S3e four-color cytometer/cell sorter. Microglia were identified based on cell surface markers (CD11b+/CD45lo) and were collected for RNA isolation. Other myeloid cells (i.e., macrophages/monocytes: CD11b+/CD45hi) were excluded from the sorted sample. Data were analyzed using FlowJo software (Ashland, OR, U.S.A.).
10
Phenotyping Testicular Immune Cells
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To detect the phenotype of TMs, testicular interstitial immune cells were incubated with flow cytometry antibodies, including FVS520 (564407, BD Biosciences, Franklin Lakes, NJ, USA), PE-F4/80 (565410, BD Biosciences), PerCP-Cy5.5-CD11b (550993, BD Biosciences), and PE-Cy7-CD86 (560582, BD Biosciences) at 4°C in the dark for 30 min. The cells were washed with PBS buffer containing 1% BSA, fixed, permeabilized, and then incubated with Alexa Fluor 647-CD206 (565250, BD Biosciences) for 30 min on ice. After that, the cells were washed and resuspended in washing buffer. Flow cytometry was performed on Agilent novoCyte (Agilent Technologies, Santa Clara, CA, USA). The fluorescence minus one (FMO) control and single staining fluorescence control of each fluorescent marker was used to assist the flow cytometry gating strategy (Supplementary Figure 2
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