Single cell suspensions of omental and blood cells were counted and stained with Zombie Aqua Fixable Viability Dye (Biolegend #423102; San Diego, CA), followed by TruStain FcX (Biolegend) to block Fc receptors. Cells were then stained with saturating concentrations of conjugated antibodies including CD45 (Biolegend #103106), CD3 (Bio-legend #100322), CD8 (Biolegend #100723), MHCII (Biolegend #107643), F4/80 (Biolegend #123130), B220 (Biolegend #103210), CD69 (Biolegend #104512), and CD103 (Biolegend #121430). Results were obtained from multiparameter flow cytometry using an Attune NxT Flow Cytometer (ThermoFisher Scientific) and analyzed with FlowJo, LLC software (Becton, Dickinson and Company, Ashland, OR: 2019.) Cells were gated via general immune cells (CD45+), lymphocytes (CD45 + CD3+), CD8 T cells (CD45 + CD3 + CD8+), CD8− T cells (CD45 + CD3 + CD8−), activated CD8 T cells (CD45 + CD3 + CD8 + CD69+ or CD103+), activated CD8− T cells (CD45 + CD3 + CD8-CD69+ or CD103+), B cells (CD45 + B220+), activated B cells (CD45 + B220 + MHCII+), and macrophages (CD45 + F4/80) + (see Supplemental Figs. 2 , 3 , 4 ).
Cd103
Manufactured by BD
Sourced in United States
CD103 is a laboratory equipment product manufactured by BD. It is designed for the detection and analysis of specific cell surface markers. The device utilizes flow cytometry technology to provide accurate and reliable data on cell populations of interest.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BD (8 mentions)
Siglec f, by BD (7 mentions)
Ifn γ, by Thermo Fisher Scientific (7 mentions)
Golgi stop reagent, by BD (4 mentions)
Il 17a, by BD (4 mentions)
Tnf α, by BioLegend (3 mentions)
Ifn γ, by BD (3 mentions)
Mhcii, by BD (3 mentions)
Cd49d l25, by BD (2 mentions)
Unlabeled cd28 l293, by BD (2 mentions)
Eomesodermin wd1928 pe efluor610, by Thermo Fisher Scientific (2 mentions)
S1pr1 sw4gypp efluor660, by Thermo Fisher Scientific (2 mentions)
Mip 1β, by BD (2 mentions)
T bet, by BioLegend (2 mentions)
Staphylococcal enterotoxin b, by Merck Group (2 mentions)
Interleukin 2 (il 2), by BioLegend (2 mentions)
Ionomycin, by BD (2 mentions)
Jagged1, by BioLegend (2 mentions)
Jagged2, by BioLegend (2 mentions)
Dnase 1, by Merck Group (2 mentions)
26 protocols using cd103
1
Flow Cytometry Analysis of Immune Cells
2
Multiparametric Flow Cytometry Assay
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The following monoclonal antibodies were used in flow cytometry: CD8 (SK1: APC-H7), CCR7 (3D12: PE-Cy7), CD69 (L78: PE), IFN-γ (B27: PE-Cy7), MIP-1β (D21-1351: PerCP-Cy5.5), from BD Biosciences (San Jose, CA); CD103 (Ber-ACT8: Ax488), TNF-α (MAb11: BV605), IL-2 (MQ1-17H12: BV650), CD107a (H4A3: BV711), CD4 (RPA-TA: BV570, BV605), CD45RO (UCHL1: BV785), CD27 (O323: BV650), CD3 (UCHT-1: Ax700), and T-bet (4B10: BV711) from Biolegend (San Diego, CA). Unlabeled CD28 (L293), and CD49d (L25) were from BD Pharmingen (San Diego, CA). Eomesodermin (WD1928: PE-eFluor610) and S1PR1 (SW4GYPP: eFluor660) were from ThermoFisher (Waltham, MA). The HIV-1 Gag peptide pool (p55, HXB2 sequence) consisted of 15-mers with an 11 amino acid overlap (BD Biosciences). Staphylococcal enterotoxin B (SEB) was from Sigma Aldrich.
3
Fluorophore-conjugated antibodies for flow cytometry
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Fluorophore-conjugated murine antibodies were purchased from eBioscience, including antibodies specific for CD8a (53–6.7; catalog #48-0081-80), Thy1.1 (HIS 51; 25–0900-82), Tim3 (RMT3-23; 14–5870-81), Lag3 (ebioC9B7W; 12–2231-81), CD137 (17B5; 17-1370-80), KLRG1 (2F1; 175893–81), and CD25 (PC 61.5; 17–0251-81). Other antibodies to FasL (K10; 106805), vβ9 (MR10-2; 553201), CD69 (H1.2F3; 104502), CD127 (A7R34; 135013), CD80 (16-10A1; 553768), CD86 (GL-1; 105011), IL-2 (JES6-5H4; 503807), IFNγ (XMG1.2; 505809), TNFα (Mab11; 502913), Bax (6A7; 633801), and Bcl-2 (10C4; 633507) were purchased from Biolegend. 7AAD (A1310) and other antibodies were purchased from BD Biosciences, including Fas (Jo2; 563647), Thy1.2 (53–2.1; 561616), CD103 (M290; 557495), purified rat anti-mouse CD16/CD32 (2.4G2; 12–4875-80), PD-1 (J43; 11–9985-81), and Vβ8.1/8.2 (553185). Antibodies against pAKT (S473; D9E; 5315S) were purchased from Cell Signaling Technology. Anti-Eomes (DAN11 mag; 12–4875-80) and the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34957) were purchased from ThermoFisher. For FasL detection, Panc1 cells were treated for 24 h with 3 µg/ml Batimastat (BB-94; ab146619) purchased from Abcam. Flow cytometry data were acquired on a BD FACS Canto II instrument or LSR II and analyzed with Flowjo v9 software.
4
Isolation and Analysis of Murine Lung Cells
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Collagenase digestion of mouse lung tissue was previously described in 30 (link). In short, mice were euthanized by CO2 asphyxiation after which perfusion was carried out by injecting 3–5 mL of phosphate buffered saline (PBS) through the right ventricle of the heart. Lungs were resected, minced with scissors, and digested in complete DMEM containing 15 mg/mL collagenase A (Roche, Indianapolis, IN), and 2500 units of DNase I (Sigma-Aldrich, St. Louis, MO) for 30 m at 37°C. Digested tissue was disrupted through a 10 mL syringe, filtered through a 100 μM pore size nytex screen and centrifuged through a 20 % Percoll solution in serum free media. 10x106 cells were then stained with appropriately diluted fluorophore-conjugated antibodies for 30 m. For intracellular cytokine staining (ICS) cells were diluted to 1x106/mL and stimulated for 4 h with PMA (10 ng / mL), ionomycin (10 μM), and Golgi-stop reagent (BD Bioscience, San Jose, CA). Antibodies used in this study are as follows: anti-CD45, CD11b, CD4, IL-17A, MHCII (I-Ab), CD103, SiglecF (BD Bioscience, San Jose, CA), CD11c, IFNγ (eBioscience San Diego, CA), DLL1, DLL4, Jagged1, Jagged2, and CD64 (Biolegend, San Diego, CA).
5
Comprehensive Immune Profiling by FACS
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Fluorescence-activated cell sorting (FACS) analysis was carried out as described previously30 (link). Briefly, cells in the SVF were suspended in FACS buffer and incubated with anti-mouse CD16/CD32 (93; Biolegend, San Diego, CA) for 15 min. Then, the cells were rinsed and resuspended in FACS buffer and stained with anti-CD11c (HL3; Biolegend) and anti-CD11b (M1/70: Biolegend) antibodies for DC and macrophages, respectively. The expression of co-stimulatory molecules was determined using mAbs to CD80 (16-10A1; BD), CD86 (GL1; BD), CD40 (3/23; BD), and MHC class II (M5/114.15.2; BD). The expression of other surface antigens was analyzed by using mAbs to F4/80 (BM8; Biolegend), CD103 (M290; BD), CD205 (NLDC145; MACS), CD206 (MR5D3; AbD Serotec), mPDCA1 (JF05-1C2.4.1:MACS), and B220 (RA3-6B2; Biolegend). To detect lipid, cells were first stained with 1 μg/ml Nile red (Wako Pure Chemicals, Osaka, Japan) for 15 min, and then analyzed with FACSVerse (BD Biosciences). The expressions of Treg related molecules were determined by using mAbs to CD25 (PC61; Biolegend), FR4 (12A5; Biolegend), CTLA4 (UC10-4B9; Biolegend) and GITR (DTA-1; Biolegend).
6
Lung mononuclear cell isolation
7
Generating Dendritic Cells from Bone Marrow
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BM‐derived DCs were isolated and induced differentiation as previously described.28 BM mononuclear cells were separated and cultured with 20 ng/mL recombinant rat granulocyte‐macrophage colony‐stimulating factor (GM‐CSF; Biovision, USA) and 20 ng/mL recombinant rat interleukin 4 (IL4; Biovision, USA) for 5 days to induce immature dendritic cells (iDCs), which were assessed by flow cytometry. iDCs were induced at day 5 with 200 ng/mL TNF‐α (PEPROTECH, MU, USA) stimulation for another 2 days to became mature dendritic cells (mDCs). Flow cytometry analysis was performed to evaluate the DCs maturation with CD11c (Abcam), CD68 (Novus), CD103 (BD), CD11b (Novus), CD86 (BD) and CD80 (Biolegend).
CD103+ DCs sorted from mDCs were cultured with or without MSC‐CM (10:1) for 48 h, and the expression of surface markers (including CD80 and CD86) on CD103+ DCs was analysed.
CD103+ DCs sorted from mDCs were cultured with or without MSC‐CM (10:1) for 48 h, and the expression of surface markers (including CD80 and CD86) on CD103+ DCs was analysed.
8
Comprehensive Immune Cell Profiling
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We used the following monoclonal antibody clones: CD103 (PE, M290, BD); CD45 (APC-Cy7, APC and FITC, 30-F11, BD); F4/80 (PE-Cy7, BM8, BioLegend); CD11c (BV421 and BV605, N418, BioLegend); CD64 (PE, X54-5/7.1, BioLegend); MHC class II (APC and PE-Cy7, M5/114.15.2, BioLegend); Ly6C (PerCP-Cy5.5, AL-21, BD); and CD11b (APC and PE, M1/70, BD). Unspecific Fc receptor binding was blocked using human immune globulin, 10% liquid (Privigen) diluted 1:66. We determined absolute cell numbers by adding fixed numbers of CaliBRITE APC-beads (6 µm) (BD Biosciences) before measurement as internal reference, excluding dead cells with Hoechst-33258 (Invitrogen). Additionally, muscularis was weighed before digestion and cell numbers were normalised to 100 mg muscularis. Flow cytometry was performed on a BD LSRFortessa II (cytometer configuration is listed on https://cores.ukb.uni-bonn.de/fccf/wp-content/uploads/sites/11/2017/05/Filter-und-Fluorochrome-Fortessa-BUV.pdf ) and data were analysed with Flow-Jo software (Tristar). Experiments including fluorescence activated cell sorting were performed on a BD Aria with a 70 µm nozzle (configuration: https://cores.ukb.uni-bonn.de/fccf/wp-content/uploads/sites/11/2017/02/Filter-und-Fluorochrome-Aria.pdf ).
9
Multiparametric Flow Cytometry of Tumor Cells
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Single tumor cell suspensions were generated by manual disruption. Two subsets of anti-human antibodies were used. Subset 1: CD45, CD3, CD25, CD69, CD62L, CD45RA, CD14, CD16, CD19 (all BD Biosciences), CD8+ (eBioscience), CD4+ and CD197 (both BioLegend). Subset 2: CD45, CD3, CD25, CD45RA, CD103, CD127, CD279, FoxP3 (all BD Bioscience), CD8+ (eBioscience) and CD4+ (BioLegend). Dead cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen). Samples were processed on an LSRFortessa flow cytometer (BD Biosciences) and data was analyzed as whole percentage of live singlet cells using FlowJo software 7.
10
Comprehensive Lung Leukocyte Profiling
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After total lung leukocyte preparation was performed, nonspecific Fc binding was blocked with a CD16/32 antibody. Subsequently, primary antibodies were added to cell samples and incubated for 30 minutes in the dark at 4°C. Primary antibodies used were anti-CD45, CD11b, CD4, IL-17A, MHCII (I-Ab), CD103, SiglecF (BD Bioscience, San Jose, CA), CD11c, IFNγ (eBioscience San Diego, CA), CD64 (Biolegend, San Diego, CA). For intracellular cytokine staining, cells were first incubated with PMA (10 ng / mL) plus ionomycin (10 μM), with the addition of Golgi-stop reagent (BD Bioscience, San Jose, CA) for 4 h at 37°C. Staining was then performed as above.
T-Distributed Stochastic Nearest-neighbor Embedding (tSNE) was implemented as a plugin in Flowjo v 10.4. Briefly, flow cytometry data for individual samples was concatenated into a single FCS file and a pre-gating strategy was implemented to rid the dataset of cell debris and doublets. Cleaned data was further gated as being Thy1.2+, IL-17+ and the tSNE algorithm was run to separate groups based on the indicated surface markers using the following parameters: perplexity 35, learning rate 1500 for 3000 iterations.
T-Distributed Stochastic Nearest-neighbor Embedding (tSNE) was implemented as a plugin in Flowjo v 10.4. Briefly, flow cytometry data for individual samples was concatenated into a single FCS file and a pre-gating strategy was implemented to rid the dataset of cell debris and doublets. Cleaned data was further gated as being Thy1.2+, IL-17+ and the tSNE algorithm was run to separate groups based on the indicated surface markers using the following parameters: perplexity 35, learning rate 1500 for 3000 iterations.
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