Spleens were harvested from naive mice and septic mice at days 7 and 14. Single-cell suspensions were created by passing cells through 70-μm pore-sized cell strainers (BD Falcon, Durham, NC). Erythrocytes were then lysed using ammonium chloride lysis buffer and washed twice using phosphate-buffered saline. Cells were stained with the following antibodies for flow cytometric studies: PE Cy7 anti-CD11b, PB anti-Ly6C, APC Cy7 anti-Ly6G, PerCpCy5.5 anti-MHCII, FITC anti-F4/80, and APC anti-CD11c (BD Pharmigen, Billerica, MA). Sytox Blue (Invitrogen, Carlsbad, CA) was used for cell viability analysis, and samples were acquired and analyzed using a LSRII flow cytometer (BD Biosciences) and FACSDiva (BD Biosciences, San Jose, CA).
Anti cd11c apc
Manufactured by BD
Sourced in United States
Anti-CD11c-APC is a fluorochrome-conjugated monoclonal antibody that recognizes the CD11c antigen, which is expressed on the surface of dendritic cells and macrophages. This product can be used for the identification and enumeration of these cell populations in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd86 pe, by BD (4 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Anti cd3 fitc, by BD (3 mentions)
Anti cd4 pe, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Anti cd80 fitc, by BD (3 mentions)
Anti b220 pe, by BD (3 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Anti cd80 pe, by BD (3 mentions)
Anti cd11b pe cy7, by BD (2 mentions)
Facsdiva, by BD (2 mentions)
Pe anti f4 80, by BD (2 mentions)
Anti cd45 fitc, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd14 fitc, by BD (2 mentions)
Anti cd11b apc cy7, by BD (2 mentions)
Anti cd4 fitc, by BD (2 mentions)
Anti cd11b fitc, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Collagenase type ia, by Merck Group (2 mentions)
39 protocols using anti cd11c apc
1
Characterization of Splenic Immune Cells
2
Multiparameter Flow Cytometry Protocol
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The following fluorochrome-coupled versions of these antibodies were used in this study. The number in brackets indicates the manufacturer´s reference.
Purchased from BD Pharmigen: PE-anti-CD3ε (553064), PerCP-C5.5-anti-IL-17A (560666), FITC-anti-CD122 (553361), BV605-anti-Vγ2 TCR (742310), Biotin-anti-CD4 (553045), PE-anti-CD45.2 (560695), FITC-anti-Ly-6C (553104), PerCP-C5.5- anti-CD64 (561194), PE-Cy7-anti-Ly-6G (560601), APC-anti-CD11c (550261), Biotin-anti-CD11b (553309), FITC-anti-CD24 (553261), BV605-Streptavidin (563260) and PE-C7-Streptavidin (557598). From eBioscience/Invitrogen: PerCP-eFluor710-anti-TCRγ/δ (46-5711-82), APC-anti-CD27 (17-0271-82), PE-Cy7-anti-TCR Vγ2 (25-5828-82), APC-anti-RORγt (17-6988-82), and Biotin-anti-CD8a (13-0081-85). From Biolegend: BV421-anti-CD44 (103040), BV421-anti-TCRγδ (118119), PE/Cy7-anti-CD27 (124216), APC-anti-CD45RB (103320), APC-anti-CD73 (127210) and BV421-Streptavidin (405225). From Miltenyi Biotec: APC-anti-IFNγ (130-120-805).
Purchased from BD Pharmigen: PE-anti-CD3ε (553064), PerCP-C5.5-anti-IL-17A (560666), FITC-anti-CD122 (553361), BV605-anti-Vγ2 TCR (742310), Biotin-anti-CD4 (553045), PE-anti-CD45.2 (560695), FITC-anti-Ly-6C (553104), PerCP-C5.5- anti-CD64 (561194), PE-Cy7-anti-Ly-6G (560601), APC-anti-CD11c (550261), Biotin-anti-CD11b (553309), FITC-anti-CD24 (553261), BV605-Streptavidin (563260) and PE-C7-Streptavidin (557598). From eBioscience/Invitrogen: PerCP-eFluor710-anti-TCRγ/δ (46-5711-82), APC-anti-CD27 (17-0271-82), PE-Cy7-anti-TCR Vγ2 (25-5828-82), APC-anti-RORγt (17-6988-82), and Biotin-anti-CD8a (13-0081-85). From Biolegend: BV421-anti-CD44 (103040), BV421-anti-TCRγδ (118119), PE/Cy7-anti-CD27 (124216), APC-anti-CD45RB (103320), APC-anti-CD73 (127210) and BV421-Streptavidin (405225). From Miltenyi Biotec: APC-anti-IFNγ (130-120-805).
3
Synthesis and Characterization of 15dPMJ₂ Derivatives
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15dPMJ2 and neutral-15dPMJ2 were synthesized as described previously [13 (link)]. GSK2606414 was purchased from Millipore Sigma (Burlington, MA, USA). The reagents, 2-mercapthoethanol, propidium iodide and tetradecanoylphorbol acetate (TPA), were purchased from Sigma-Aldrich (St. Louis, MO, USA). GAPDH antibody was obtained from Cell Signaling Technologies (Beverley, MA, USA). Anti-CHOP10 was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-Glo® 3/7 assay kit, CellTiter-Glo 2.0 assay kit, and MTS reagent were purchased from Promega Life Sciences (Madison, WI, USA). Anti-calreticulin (Alexa Fluor 647) was from Abcam (Cambridge, MA, USA). HMGB1 ELISA kit was purchased from Novatein Biosciences (Woburn, MA, USA). Recombinant mouse GM-CSF, 7-AAD, PE-Cy7-anti-CD80, APC-Cy7-anti-CD86, PE-Anti-Hsp90 and FITC-Anti-Hsp70 were obtained from BioLegend (San Diego, CA, USA). APC-anti-CD11c and mouse Fc block were purchased from BD Biosciences (San Jose, CA, USA). FITC-anti-MHC class II, anti-CRT and CellTracker Green CMFDA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). IRDye 800CW anti-rabbit and IRDye 680RD anti-mouse secondary antibodies were from LI-COR Biosciences (Lincoln, NE, USA).
4
Immunophenotyping of Blood Mononuclear Cells
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Single-cell suspensions of blood samples were washed with ice-cold PBS for three times and subsequently treated with FCM lysing solution for 15 min. Then, cells were washed with ice-cold PBS to remove the excess FCM lysing solution and stained with PE-anti-F4/80, FITC-anti-CD45, FITC-anti-CD3, APC-anti-CD11c, and PE-anti-CD4 antibodies from BD Bioscience (San Diego, USA) in the dark at RT for 15 min. The data were analyzed with FlowJo software (San Carlos, CA). The portion of mononuclear macrophage (CD45+, F4/80+), T helper cells (CD3+, CD4+) and dendritic cells (CD45+, CD11c+) were determined by flow cytometry.
5
PBMC Multiparametric Flow Cytometry
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PBMCs were thawed and washed with RPMI 1640 followed by 1X PBS. A maximum of 106 PBMCs per sample were used for staining. Live/Dead cell exclusion was performed using AQUA Live/Dead Fixable stain (Invitrogen Life technologies, Eugene, USA). Non-specific binding sites were blocked using a Fluorescence-activated cell sorting (FACS) buffer (1X PBS, 2% hi-FBS and 0,1% sodium azide) supplemented with 20% hi-FBS and 10ug mouse IgG (Sigma-Aldrich, St-Louis, USA) per 106 cells. PBMCs were stained using the following fluorochrome conjugated mouse anti-human monoclonal antibodies: AlexaFluor 700-A anti-CD16, APC anti-CD11c, FITC anti-CD3, PE-Cy7 anti-HLA-DR, and PerCP-Cy5.5 anti-CD14 (BD-Biosciences, San Jose, CA, USA), PE anti-BLyS (ebiosciences, San Jose, CA, USA). PBMCs were fixed with 1.25% paraformaldehyde and kept at 4 °C for a minimum of 12 hours before flow cytometry analysis. A minimum of 105 events per sample were acquired with a LSRFortessa (BD-Biosciences, San Jose, CA, USA) and analyzed with FlowJo7.6.3 software (TreeStar, Ashland, OR, USA). Flow-cytometry data analysis quadrants were set based on the expression values obtained with fluorescence minus one (FMO) and isotype controls. Representative FMO staining controls can be viewed in Supplementary Fig. S3 .
6
Multicolor Flow Cytometry Immunophenotyping
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The analysis was performed by using a BD FACSCanto cytometer and BD FACSDiva software. APC anti-CD1a (# 559775), FITC anti-CD1a (#555806), BV421 anti-CD14 (#563743), FITC anti-CD14 (#555397), PE anti-CD14 (555398), PE anti-CD16 (#347617), FITC anti-CD64 (#555527), APC anti-CD209 (#551545), PE anti-CD1c (#564900), PE anti-CD86 (# 555658), FITC anti-CD80 (#560926), FITC anti-HLA-DR (#555811), APCCy7 anti-HLA-DR (#335796), APCCy7 anti-CD11b (#557754), APC anti-CD11c (#559877) were obtained from BD Biosciences. Sorting of CD1a + CD16 -cells was performed using a BD FACSCanto cytometer.
7
Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells
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For flow cytometry analysis, harvested tumors were minced into small pieces with scissors and incubated in a digestion buffer at 37 °C for 70 min. The suspensions were filtrated using a cell strainer (BD Falcon, New Jersey, USA) and centrifuged at 2000 rpm for 5 min. The collected cells were re-suspended in ACK Lysis Buffer to remove red blood cells. Next, the obtained cells were resuspended in PBS and stained by Fixable Viability Dye eFluorTM 780 (Thermo Fisher Scientific Co., Ltd., Shanghai, China) to exclude dead cells. Then, cells were washed with PBS and stained with anti-CD16/CD32 antibody (BD, New Jersey, USA) for Fc blocking to prevent nonspecific binding. Subsequently the cells were incubated with Percp-Cy5.5-anti-CD45, BV510-anti-CD3, FITC-anti-CD4, BV421-anti-CD8, PE-anti-B220, APC-anti-CD11c, FITC-anti-CD11b, PE-anti-F4/80, BV650-anti-CD86 and PE-Cy7-anti-CD206 (BD, Shanghai, China) to specifically label the T lymphocytes, B lymphocytes, DCs and Macrophage. Expect Fixable Viability Dye eFluorTM 780 and anti-CD16/32. All the above staining and centrifugation processes were carried out at 4 °C under darkness. The samples were analyzed by FCM (Sony ID7000™, Tokyo Metropolis, Japan).
8
Characterizing Lymphocyte Subsets in Liver Transplant Recipients
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The following reagents were all obtained from BD Biosciences: FITC-anti-CD3, CY5.5-anti-CD4, CY5.5-anti-CD8, PE-anti-CD19, APC-anti-CD16, PE-anti-CD56, PE-anti-CD4, FITC-anti-Lin1, PerCPCY5.5-anti-CD123, and APC-anti-CD11C. 5 mL of whole blood for ow cytometric measurement was taken from liver transplant recipients. Peripheral blood mononuclear cells (PBMC) were isolated by coll density gradient centrifugation and resuspended in phosphate-buffered saline (PBS). Then, PBMC were stained with antibodies mentioned above at 4℃ in the dark for 20 min. After that, PBMC were washed once with 2 mL PBS and resuspended in 400 µl PBS for ow cytometry analysis.
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. The lymphocytes evaluated were T (CD3 + ), TCD4 (CD3 + CD4 + ), TCD8 (CD3 + CD8 + ), B (CD19 + ), NK (CD56 + CD16 + ), NKT (CD3 + CD56 + CD16 + ) and DC (lin1 -CD11c + and lin1 -CD123 + ). Flow cytometry characterization of circulating lymphocyte subsets was presented in Suppl Fig. 1.
The absolute numbers of lymphocyte subsets were calculated using the percentages obtained in ow cytometry and the lymphocyte counts obtained in routine blood tests on the same day.
Flow cytometry was performed in NovoCyte D2060R (ACEA Biosciences Inc). NovoEXpress software (San Diego, CA, USA) was used for analysis. The lymphocytes evaluated were T (CD3 + ), TCD4 (CD3 + CD4 + ), TCD8 (CD3 + CD8 + ), B (CD19 + ), NK (CD56 + CD16 + ), NKT (CD3 + CD56 + CD16 + ) and DC (lin1 -CD11c + and lin1 -CD123 + ). Flow cytometry characterization of circulating lymphocyte subsets was presented in Suppl Fig. 1.
The absolute numbers of lymphocyte subsets were calculated using the percentages obtained in ow cytometry and the lymphocyte counts obtained in routine blood tests on the same day.
9
In Vivo Analysis of DC Maturation
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To examine DC maturation in vivo, TRAMP-C1 tumor-bearing C57BL/6 mice were treated with various strategy as described in in vivo therapy section. (n = 6) The inguinal lymph nodes were harvested 8 days post third treatment. The frequency of DC maturation in the LNs was then examined by flow cytometry after immunofluorescence staining with anti-CD11c-APC, anti-CD80-FITC, and anti-CD86-PE antibodies (BD Pharmingen™). To determine the intra-tumoral infiltration of CD8+(CD3+CD4−CD8+), lymphocytes were stained with anti-CD3-Brillant violet 510, anti-CD4-PE, anti-CD8-PE-cy7. All antibodies were diluted with the ratio of 1:100. And, signal color beads (OneComp eBeads™ Compensation Beads) and unstained bead were used for compensation. The frequency of CD8+(CD3+CD4−CD8+) lymphocytes in the LNs were also examined. Gating strategies used for cell sorting was shown in Supplementary Fig. 17 .
10
Flow Cytometry Analysis of Kidney and Spleen Cells
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Kidneys and spleens were dissected and ground separately. The kidney fragments were digested with 2 mL of collagenase type IA (2.5 U·mL−1, Sigma Chemical Company, St. Louis, MO, USA) in PBS containing 10 mM CaCl2 at 37 °C for 30 min. After being washed, the kidney and spleen slurries were passed separately through a 40-μm strainer (BD Biosciences, Franklin Lakes, NJ, USA) and then washed with PBS. Cells were collected by centrifugation at 1500 rpm for 5 min and incubated in PBS containing 2 mM EDTA and 2% FBS plus primary antibodies for 30 min at 4 °C. The cells were resuspended at 1 × 107 cells/mL before sorting or analysis. The cells were separated and analyzed on BD FACSAria II or BD FACSCanto II (BD Biosciences) by Beijing Institute of Heart, Lung and Blood, Vessel Diseases Cytometry and Cell Sorting Core Facility, and data were collected using FACSDiva 7.0 software (BD Biosciences). The antibodies used were anti-CD4-PE (Clone RM4-5), anti-CD3e-PE-cf594 (Clone 145-2C11), anti-CD8a-APC-cy7 (Clone 53-6.7), anti-CD11c-APC (Clone HL3), and anti-CD45-PerCP-cy5.5 (Clone 30-F11), all of which were purchased from BD Biosciences.
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