SPCs (Lipoid S100) were obtained from Lipoid GmbH (Ludwigshafen, Germany) with a purity of 97.6%. It was used as received and stored at −20 °C. GDO was a kind gift from Croda, UK, which contained minimally 95% diglycerides according to the producer. All tool compounds were used as obtained. IR820 was purchased from Sigma. aPD-L1(catalog number BE0101) used in vivo purchased from Bio X Cell. Anti-CD3-PerCP-Cy5.5 (catalog number 551163), anti-CD4-FITC (catalog number 553046), anti-CD8-PE (catalog number 553032), anti-CD4-FITC (catalog number 553046), anti-CD25-APC (catalog number 557192), and anti-Foxp3-PE (catalog number 563101), anti-CD11b-FITC (catalog number 557396), anti-CD11c-FITC (catalog number 557400), anti-CD80-PE (catalog number 560016), anti-CD86-APC (catalog number 553692), anti-LY-6G/LY/6C-PE (catalog number 553128), anti-CD3-FITC (catalog number 553065), anti-CD8-PErCP-Cy5.5 (catalog number 553030), anti-CD62L-APC (catalog number 562910), and anti-CD44-PE (catalog number 559250) were purchased from BD Biosciences.
Anti cd11c fitc
Manufactured by BD
Sourced in United States
Anti-CD11c-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD11c cell surface antigen. CD11c is a marker expressed on myeloid dendritic cells, monocytes, and macrophages. This antibody can be used for the identification and enumeration of these cell types in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (5 mentions)
Anti cd86 pe, by BD (4 mentions)
Anti cd4 fitc, by BD (3 mentions)
Anti foxp3 pe, by BD (3 mentions)
Anti cd11b fitc, by BD (3 mentions)
Anti cd80 pe, by BD (3 mentions)
Anti cd25 apc, by BD (3 mentions)
Anti mhcii pe, by BD (3 mentions)
Anti cd11b pe cy7, by BD (3 mentions)
Anti cd11b pe, by BD (3 mentions)
Anti cd8 fitc, by BD (3 mentions)
Anti cd3 fitc, by BD (2 mentions)
Anti cd8 pe, by BD (2 mentions)
Anti cd86 apc, by BD (2 mentions)
Anti cd62l apc, by BD (2 mentions)
Anti cd3 percp cy5, by BD (2 mentions)
Anti cd11b apc cy7, by BD (2 mentions)
Anti cd4 pe cy7, by BD (2 mentions)
Anti cd4 v500, by BD (2 mentions)
Anti cd8 v450, by BD (2 mentions)
28 protocols using anti cd11c fitc
1
Characterizing Immune Cell Responses
2
Phenotypic Characterization of Peritoneal Cells during Toxoplasma Infection
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On the third day of infection, peritoneal cells from WT mice infected with 1 × 107 Nc-1 tachyzoites were collected and stained for phenotypic characterization and Dectin-1 expression. Briefly, mice were euthanized, and their peritoneal cavities were washed with ice-cold PBS. The suspension was then centrifuged at 400 × g, at 4°C, for 10 min. The cell pellet was resuspended in PBS with 5% of normal rabbit serum, at room temperature, for 15 min, prior to incubation with the appropriate antibodies: anti-CD11b-APC-Cy7, anti-CD11c-FITC, anti-MHCII-PE, anti-CD11c-V450, anti-CD19-APC-Cy7, anti-CD3-Pacific Blue, anti-CD49b-APC (BD Biosciences), and anti-Dectin-1-PE (R&D Systems, Minneapolis, MN, USA). Cells were incubated with primary antibodies conjugated to the different fluorochromes for another 30 min, at room temperature. After washing, cells were suspended in PBS with 3% formaldehyde, read by flow cytometry (FACSCantoII, BD Biosciences), and analyzed using dedicated software (FlowJo X, Tree Star Inc., Ashland, OR, USA).
3
Multiparametric Flow Cytometry Analysis
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After blocking with anti-FcR (CD16/32, BD bioscience) for 20 min, cells were incubated with specific mAbs at 4°C for 30 min. The following mAbs were used: anti-mouse CD3e-APC-Cy7; anti-CD4-PE-Cy7; anti-NK1.1-APC; anti-CD11b-PE-Cy7; anti-CD11cFITC; 7-AAD; anti-PDCA-1-APC; anti-CCR9-PE; anti-IFN-γ-FITC; and anti-IL-17-APC (eBioscience, BD bioscience). Background fluorescence was assessed by staining with irrelevant anti-rat isotypes (BD bioscience). Stained cells were analyzed by flow cytometry (FACS Canto II, Becton Dickinson Co.) and data analyzed using FlowJo software (Tree Star Inc.) [12] (link).
4
Quantification of Gr-1+ CD11b+ Granulocytes in Infected Lungs
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To determine the absolute numbers of Gr-1+ CD11b+ granulocytes in the lungs from infected animals, flow cytometric analysis of single-cell suspensions was performed. After blocking of nonspecific binding by adding an anti-FcγRIII/II antibody (BioLegend, Amsterdam, The Netherlands) and a cocktail of mouse, rat, and hamster serum, cells were incubated with optimal amounts of the following specific antibodies (all BD Biosciences, Heidelberg, Germany): anti-CD8-V450, anti-CD4-V500, anti-CD11c-FITC, anti-CD11b-PE, anti-Ly-6G-PerCP-Cy5.5, anti-CD90.2-PE-Cy7, anti-Gr-1-APC, and anti-MHCII(IA/IE)-APC-e780. Measurement was performed on a FACSCanto™ II (BD Bioscience) and the FCS files were analyzed using the FCS Express 7 Flow Cytometry software (DeNovo™ Software, Pasadena, CA, USA). Absolute cell numbers were calculated as follows: (total cell count lung/100) × % of Gr-1+ CD11b+ cells (of analyzed flow cytometric leukocytes) = absolute cell numbers.
5
Flow Cytometry Analysis of BAL Cells
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Flow cytometry of BAL cells was performed by using the following monoclonal antibodies: anti-CD8a-AlexaFlour750 (clone 5H10, Thermo Fisher Scientific; AB_10374588), anti-Ly6c-FITC (clone AL-21, BD Biosciences, San Jose, CA USA; AB_394628), anti-CD4-PerCP-Cy5.5 (clone RM 4–5, BD Biosciences; AB_393977), anti-CD62L-PE-Cy7 (clone MEL-14, eBioscience, affymetrix, Frankfurt am Main, Germany; AB_469632), anti-CD3-eFlour450 (clone 17A2, eBiosciene; AB_1272229), anti-CD25-APC (clone PC61, BD Biosciences; AB_398623), anti-Gr1-PacificOrange (clone Rb6-8C5, Thermo Fisher Scientific; AB_2556571), anti-CD19-PE-Cy7 (clone 1D3, BD Biosciences; AB_10894021), anti-CD11b-APC-Cy7 (clone M1/70; BD Biosciences; AB_396772), anti-CD11c-FITC (clone HL 3, BD Biosciences; AB_395060), anti-CD45-AlexaFlour700 (clone 30-F11, BioLegend, San Diego, CA USA; AB_493714), anti-F4/80-APC (clone BM8, eBioscience; AB_469451) and anti-MHC-II (I-A/I-E)-PerCP-Cy5.5 (clone M5/114.15.2, BioLegend; AB_2191072) [23 (link)]. Data were acquired using a LSRII flow cytometer (BD Bioscience) and further analyzed with FACSDiva software (BD Biosciences) and FlowJo V.7.2.2 (Tree star, Ashland, USA). The gating strategy is displayed in S1 Fig .
6
Isolation and Sorting of MDSC Subsets
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We isolated Bone marrow cells (BMCs) from the tibias and femurs of the mice by flushing the bone marrow cavity with Minimum Essential Medium (α-MEM; Invitrogen). BMCs were cultured in 6-well plate at 1 × 106 cells/mL in complete medium supplemented with recombinant GM-CSF (20 ng/mL, PeproTech, Rocky Hill, NJ, USA) for 3 days. The cultured cells were harvested and stained with anti-CD11c FITC, anti-CD11b PerCP5.5, anti-Gr-1 APC, and anti-PD-L1 PE Ab (BD Biosciences) following FcR blocking (BD Biosciences). CD11c-CD11b+Gr-1+PD-L1+ and CD11c-CD11b+ Gr-1+PD-L1-MDSC subsets were sorted using a Beckman Coulter MoFlo XDP. The purity of the sorted PD-L1+MDSC and PD-L1MDSC was > 95%.
7
Purification and Characterization of Primitive Blood Cell Populations
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CD34+ cells from ABM and UCB were enriched using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to manufacturer instructions and our previous reports [9 (link)]. After staining with PE-conjugated antilineage markers (CD3, CD8, TCR, CD56, CD14, CD11b, CD20, CD19, and CD235a), anti-CD34-APC, anti-CD38-FITC, and anti-CD45RA-PE-TxR conjugated antibodies, primitive cell populations were highly purified by multicolor flow cytometry using a FACSAria sorter (BD Biosciences). Hematopoietic stem cells (HSC) were separated as Lin−CD34+CD38−CD45RA−, while multipotent progenitors (MPP) were sorted as Lin−CD34+CD38+CD45RA−, and early lymphoid progenitors (ELP) as Lin−CD34+CD38+CD45RA+, as described [9 (link)]. Upon harvesting from culture, anti-CD56-PE, anti-CD11c-FITC, and anti-CD16-APC (BD Biosciences) were used to evaluate innate cell production in lymphoid lineage conditions. NK cells were identified by flow cytometry in a FACSCanto II equipment (BD Biosciences) as CD56+CD11c+ or CD56+CD16+CD11c+ cells, while dendritic cells (DC) were detected as CD56−CD11c+. Analysis of flow cytometry data was performed using the FlowJo 10 software (TreeStar Inc., USA).
8
Immunophenotyping of Tumor-Infiltrating Cells
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Tumor samples were digested using half the amount of reagent required per sample of Miltenyi Biotech Liver Kit as per the manufacturer's instructions in C-tubes on a gentleMACS™ dissociator (Miltenyi Biotech) with heaters. After digestion, samples were filtered through Falcon filter cap tubes and washed with 2 ml Dulbecco's modified Eagle medium (Gibco). Samples were centrifuged at 500 g for 5 min, washed twice with AutoMACS Rinsing Solution (Miltenyi Biotech) with 1% bovine serum albumin and 0.5 mM EDTA, and resuspended in 1 ml of the aforementioned solution for counting and viability check on an Attune NxT cytometer. Cells (106) were incubated with the following antibodies: anti-CD16/32 APC-R700, anti-CD11b APC, anti-F4/80 PE-Cy7, anti-EpCAM PerCP-Cy5.5, anti-CD45 BV605, anti-CD11c FITC, anti-NKp46 PE (BD Biosciences; diluted at 1:000 to 1:10.000). Data were acquired on a BD Biosciences FACSymphony A5 cytometer and analyzed with FlowJo Software (BD Biosciences).
9
Isolation and Characterization of Kidney Dendritic Cells
10
Pullulan-based Immunotherapeutic Delivery
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Pullulan was purchased from Meihua Group (Hebei, China). PAA (average MW of 450,000) was purchased from Aladdin Industrial Corporation Chemical (Shanghai). N-(3-Dimethylaminopropyl)-N’-Ethylcarbodiimide Hydrochloride (EDC) was purchased from Heowns Chemical (Tianjin, China). 4-Dimethylaminopyridine (DMAP) and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd. Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. aCD47 (Catalog no. BE0270) used in vivo was purchased from BioX Cell. AF790-labeled goat anti-mouse IgG (H&L) (Catalog no. 115-655-146) was purchased from Jackson ImmunoResear. Anti-CD3-PerCP-Cy5.5 (Catalog no. 551163), anti-CD4-APC (Catalog no. 553051), anti-CD8-FITC (Catalog no. 553030), anti-CD86-PE (Catalog no. 553692), anti-CD80-APC (Catalog no. 560016), anti-Foxp3-PE (Catalog no. 563101), anti-CD11c-FITC (Catalog no. 557400), anti-CD62L-BV421 (Catalog no. 562910), anti-CD44-APC (Catalog no. 559250) and anti-CD11b-PE-Cy7 (Catalog no. 552850) were purchased from BD Biosciences. Anti-mouse CD206-PE (Catalog no. 141705) and anti-mouse F4/80-FITC (Catalog no. 123107) were purchased from Biolegend. Human IgG ELISA Quantitation Set (Catalog no. E80-104) was purchased from Bethyl Laboratories, Inc.
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