Human cervical tissue was obtained from two sets of five healthy women (age range 42–47 years old) undergoing hysterectomy for benign indication at HUGTP. After confirmation of healthy tissue status by the Pathological Anatomy Service, a piece from ecto and endocervix separated by anatomical localization was delivered to the laboratory in refrigerated RPMI 1640 medium (Cellgro, Manassas, VA) containing 10% FBS (Lonza, Basel, Switzerland), 500U/mL penicillin, 500μg/mL streptomycin, 5μg/mL fungizone and 1μg/mL gentamycin (Life Technologies). Tissue was processed within the next 12 h after surgery, and 8-mm3 block-dissection was performed as described [26 (link)]. Tissue digestion of 5–9 pieces of ecto or endocervix with collagenase IV (Invitrogen) was immediately executed as described [26 (link)]. Tissue blocks were then dissociated manually with a disposable pellet pestle and filtered through a 70μm-cell strainer (BD Biosciences). After centrifugation, pellet was suspended in staining buffer (1% mouse serum, 1% goat serum in PBS) and stained with different combinations of CD3-eFluor 605 (eBiosciences), CD14-V450, CD11c-PE-Cy7, CD8-V500, HLA-DR-PerCP-Cy5.5, CD69-Horizon PE-CF594 (BD Biosciences), NK1.1-PE, γδTCR-FITC, Vα7.2-APC-H7 (Miltenyi Biotec), CD103-FITC and Vα24-APC (BioLegend). Data were acquired and analyzed as described for blood.
Cd103 fitc
Manufactured by BioLegend
Sourced in United Kingdom, United States
CD103-FITC is a fluorescently labeled antibody that binds to the CD103 antigen. CD103 is a cell surface protein that is expressed on a subset of T cells and dendritic cells. The FITC label allows for the detection and identification of CD103-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Cd11c pe cy7, by BD (2 mentions)
Hla dr percp cy5, by BD (2 mentions)
Cd3 efluor 605, by Thermo Fisher Scientific (2 mentions)
Cd86 apc, by BioLegend (2 mentions)
Cd11c pe cy7, by BioLegend (2 mentions)
Cd14 v450, by BD (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Cd8 v500, by BD (1 mentions)
Nk1.1 pe, by Miltenyi Biotec (1 mentions)
γδ tcr fitc, by Miltenyi Biotec (1 mentions)
Facsaria 1, by BD (1 mentions)
Cd3 pe, by BD (1 mentions)
Cd11c apc, by BioLegend (1 mentions)
9 protocols using cd103 fitc
1
Multiparametric Phenotyping of Cervical Immune Cells
2
Immunophenotyping of Hairy Cell Leukemia
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HCL samples were stained for CD19-eFluor450 (eBioscience, Hatfield, UK)/CD19-V450 (BD), CD11c-APC (BioLegend, London, UK), CD103-FITC (BioLegend/BD), CD27-APC-eFluor780 (eBioscience) and CD3-PE (BD/eBioscience) as described for immunophenotyping. HCLc tumour cells were sorted and selected as a CD19+CD11cHICD103+CD27- population and germline T cells from the same sample as CD3+ using the FACS Aria I (BD) (Fig 1 ). Sorted populations were re-analysed for purity on the FACS Aria I: purity of HCLc sorts was 92.9–98.3% and of T cells 96.7–100% (Fig 1 is a typical example). Data were analysed using FACS Diva (BD) and FlowJo (TreeStar).
3
Differential Immunophenotyping of NK Cell Subsets
4
Multicolor Flow Cytometry Immunophenotyping
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Intestinal lamina propria cells and MLN cells were labeled with CD4-FITC, CD25-APC (eBioscience), CD8-APC-Cy7, CD3-PerCP (Biolegend), and B220-BV570 (BD Biosciences). Labeling of intracellular FoxP3 was performed after extracellular staining and fixation/permeabilization of cells. GM-CSF-derived dendritic cells were labeled with CD11b-PerCP Cy5.5 (BD Biosciences), CD11c-PE-Cy7, I-A/I-E-APC-Cy7, CD80-PE, CD86-APC, CD8-FITC, and B220-BV570 (Biolegend). Flt3L-derived dendritic cells were labeled with CD11c-PE-Cy7, CD11b- or Siglec-H-PerCP Cy5.5 (Biolegend), I-A/I-E-APC-Cy7, CD317-PE, CD40-Alexa Fluor 647, CD103-FITC, and B220-BV570. Coculture T cells were labeled with CD4-PerCP Cy5.5 (BD Biosciences), CD127-PE-Cy7, CD73-APC, CD195 (CCR5)-FITC, CD62L-BV570, and CD25-APC-Cy7 (Biolegend).
5
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.
6
Phenotypic Analysis of Influenza-Specific T Cells
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Mononuclear cells were washed twice with cold PBS with 0.5% FBS and 0.1% NaN3 and centrifuged at 1,600 rpm for 5 min at 4°C. Purified rat anti-mouse CD16/CD32 (BD Pharmingen, San Diego, CA, USA) Fc blocker and 5 µg/ml streptavidin (Invitrogen) were added to each sample and incubated for 20 min at 4°C. The following cocktail was used for surface staining: CD3-APC/Cy7 (Clone 17A2; BioLegend), CD8-PE (Clone KT15; MBL Life science, Tokyo, Japan), CD62L-Alexa Fluor 700 (Clone MEL-14; BioLegend), CD69-PE/Cy7 (Clone H1.2F3; BioLegend), CD103-FITC (Clone 2E7; BioLegend) and influenza B-NP specific Dd/NP166-174 (FSPIRITFL) tetramer. The B-NP-specific Dd/NP166-174 (FSPIRITFL) tetramer was produced as described previously (20 (link)). In brief, recombinant Dd protein was refolded in the presence of NP166-174 peptide and β2m, biotinylated with biotin-protein ligase BirA, and then purified with Superdex-75 gel filtration column (GE Healthcare Life science, Chicago, IL, USA). The purified monomer was tetramerized with Alexa Fluor 647-streptavidin conjugates (Thermo Fisher Scientific, Waltham, MA, USA). Stained cells were washed, fixed using BD FACS Lysing Solution (BD Pharmingen) at RT for 20 min, and collected on an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed using Flowjo software (TreeStar Inc., Ashland, OR, USA).
7
Comprehensive T-cell Phenotyping by Flow Cytometry
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Single-cell suspension (106 cells/tube) was incubated with anti-CD16/32 antibody to block Fc receptors for 15min at 4°C. For surface staining, the cells were stained with antibodies or matched isotype control for 20 min at 4°C in the dark. The antibodies included anti-mouse CD3-PECY7, CD4-FITC, CD8-Percpcy5.5, CD25-APC, CD11c-PE, CD86-APC, CD80-PerCP-eFlour710, PD-L1-PECY7, MHCII-Alexa Flour 700, MHCII- APC, and CD103-FITC antibodies (all from Biolegend, San Diego, CA, USA). For intracellular staining, cells were cultured in the presence of 2μl/ml cell stimulation cocktail (eBioscience) for 6 h. The cells were fixed and permeabilized using fixation and permeabilization solutions, then stained with anti-mouse Foxp3-PE, IL4-PE, IFN-γ-APC, IL10-PE antibodies (all from eBioscience), or isotype control for 30 min at 4°C in the dark. The cells were detected using BD FACSVerse (BD Biosciences, Franklin Lakes, NJ, USA) or Aurora (Cytek, Fremont, CA, USA), and data were analyzed using the FlowJo 10.0.7 software (Tree Star, Ashland, OR, USA).
8
Murine Immune Cell Characterization
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The reagents and antibodies used in this study were referenced from our previous reports28 (link)–30 (link),32 (link),60 (link). Lipopolysaccharide (LPS) from Escherichia coli (LPS O55:B5, L2880) was purchased from Sigma-Aldrich (MO, USA). Pam3csk4, CpG-ODNs and cGAMP (cyclic guanosine monophosphate–adenosine monophosphate) were purchased from Invivogen (CA, USA). Murine GM-CSF (granulocyte–macrophage colony-stimulating factor) was purchased from Peprotech (Rocky Hill, NJ, USA). The anti-mouse TLR7 mAb A94B10 and the anti-TLR9 mAb NaR9 were purified from ascitic fluid, as reported previously29 (link),30 (link). Streptavidin–phycoerythrin (PE), anti-mouse IgG1-PE, anti-mouse IgG2a-PE, isotype control antibodies (mouse IgG1, mouse IgG2a), and antibodies against mouse CD16/32, CD3-PE-Cy7, CD45-APC-Cy7, B220-FITC, CD11b-APC, CD11c-BV421, CD11c-PE-Cy7, Siglec-F-FITC, Ly-6G-FITC, CD4-BV510, CD8α-Percp/Cy5.5, CCR3-Percp/Cy5.5, I-A/I-E-BV510, CD103-FITC and IL17A-APC were purchased from BioLegend (San Diego, CA, USA). Anti-mouse IL5-PE, anti-mouse IL-2-APC, and anti-mouse IL13-eFluor450 were purchased from Invitrogen (Thermo Fisher Scientific, Minato-ku, Tokyo, Japan). Mouse IL-17A and IL-2 recombinant proteins were purchased from BioLegend.
9
Generating Single-Cell Suspensions from Tumor
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To generate a single-cell suspension of the tumor for cell sorting, tumors were digested in enzyme medium on a shaker for 30 min at 37°C and then successively filtered through 140, 100 and 40 μm strainers. Enzyme medium comprised RPMI-1640 (ThermoFisher Scientific) supplemented with trypsin inhibitor (Sigma-Aldrich) at 1 mg/ml, hyaluronidase (Sigma-Aldrich) at 0.3 mg/ml, collagenase (Sigma-Aldrich) at 1 mg/ml and DNase (Pulmozyme) at 10 U/ml. Fresh tumor single-cell suspensions were labeled with the fluorescent-labeled antibodies CD3-AF700 (clone: SK7), CD4-APC Fire750 (clone: SK3), CD8-BV785 (clone: SK1), PD-1-AF647 (clone: EH12.2H7), CD103-FITC (clone: Ber-ACT8) and CD39-BV421 (clone: A1) (all from BioLegend). Populations of interest were purified using a FACSAria II based on PD-1, CD39 and CD103 expression for single-cell sequencing and in vitro expansion using excess irradiated (5,000 rad) allogeneic PBMC feeder cells pooled from two different donors in 50/50 T-cell medium supplemented with 30 ng/ml anti-CD3 antibody (OKT3, Biolegend) and 3,000 IU/ml IL-2. 50/50 media consisted of CM and AIM-V media at a ratio of 1:1. After about 2 weeks, T cells were used in co-culture assays or cryopreserved for further analysis.
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