Degarelix (as acetate), a third generation LHRH-Ant (Firmagon), was resuspended in sterile water for injection and administered s.c. to mice at a dose of 40 µg/g. Lupron (11.25-mg 3-mo depot), an LHRH-Ag, was prepared according to the manufacturer’s instructions and administrated intramuscularly to mice at a dose of 20 µg/g. Degarelix and Lupron were purchased from the MSKCC Pharmacy. Testosterone propionate (Sigma-Aldrich) was resuspended in peanut oil and injected daily s.c. (1 mg/mouse) in 100 µl. Surface antibodies against CD44 (IM7), EpCAM (G8.8), PDGFRa (APA5), PECAM-1 (390), CD45 (30-F11), and H-2Kk (AF3-12.1.3) were purchased from eBioscience; anti-Ly-51 (BP-1), CD34 (RAM34), CD62L (MEL-14), H-2Kb (AF6-88.5), IFN-γ (XMG1.2), IL-2 (JES6-5H4), c-Kit (2B8), CD3ε (145-2C11), CD25 (PC61), TER-119 (TER-110), and CD8α (53–6.7) were purchased from BD; anti-CD4 (RM4-5) and B220 (RA3-6B2) were purchased form Invitrogen; anti-CD44 (IM7), CD90.2 (30-H12), and IA/IE (M5/114.15.2) were purchased from BioLegend; and Ulex europaeus agglutinin 1 (UEA-1) was purchased from Vector Laboratories. For in vitro cultures of T cell differentiation (Fig. 2, A and B ), cells were stained with antibodies to lineage (Lin)-specific markers: CD8, CD11c, NK1.1, CD3, CD4, B220, CD11b, and GR-1.
I a 1 e m5 114
Manufactured by BioLegend
Sourced in United States
The I-A/I-E (M5/114.15.2) is a laboratory reagent used for the detection and analysis of major histocompatibility complex (MHC) class II molecules. It is a monoclonal antibody that binds to the I-A and I-E regions of the MHC class II complex in mice. This product can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and ELISA, to identify and characterize cells expressing these MHC class II molecules.
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Ly6g 1a8, by BioLegend (3 mentions)
Siglecf e50 2440, by BD (3 mentions)
Fortessa, by BD (3 mentions)
F4 80 bm8, by Thermo Fisher Scientific (3 mentions)
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Viability 65 0863 14, by Thermo Fisher Scientific (3 mentions)
B220 ra3 6b2, by BioLegend (2 mentions)
Ebio13a, by Thermo Fisher Scientific (2 mentions)
Tc11 18h10, by BD (2 mentions)
Ly6c hk1, by BioLegend (2 mentions)
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Cd45 30 f11, by BioLegend (2 mentions)
Cd19 6d5, by BioLegend (2 mentions)
Cd4 gk1, by BD (2 mentions)
Cd11c n418, by BD (2 mentions)
Cd45.1 a20, by BioLegend (2 mentions)
11 protocols using i a 1 e m5 114
1
Modulating Androgen Levels in Mice
2
Immune cell antibody staining
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Mouse antibodies (Abs) and isotype control Abs (IgG1, IgG2a, and IgG2b), anti-CD3 (17A2), CD4 (GK1.5), CD8α (53–6.7), tumor necrosis factor (TNF)-α (MP6-XT22), perforin (S16009A), CD11c (N418), CD40 (3/23), CD80 (16-10A1), CD86 (GL-1), anti-interferon (IFN)-γ (B27), MHC class I (H2Kd, 28-8-6), and MHC class II (I-A/I-E, M5/114.15.2) were purchased from BioLegend (San Diego, CA, USA).
3
Isolation and Characterization of Murine Immune Cells
4
Immune Cell Phenotyping Protocol
5
Multiparameter Flow Cytometry Analysis
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Single cell splenocyte suspensions were prepared and stained in ice-cold PBS with Ghost Dye Violet 510 (Tonbo Biosciences, San Diego, CA) to exclude dead cells. Surface staining was subsequently performed in PBS containing 3% FCS and 5 mM EDTA in the presence of unconjugated anti-CD16/32 clone 2.4G2 (purified in-house). Antibody clones used for staining were directed against CD45R (RA3-6B2), CD23 (B3B4), CD19 (1D3), CD11c (HL3) and TCRß (H57-597) (BD Biosciences, Franklin Lakes, NJ); CD62L (Mel-14), CD138 (281–2), CD11b (M1/70), TCRß (H57-597) and I-A/I-E (M5/114.15.2) (BioLegend, San Diego, CA); SiglecH (eBio440c) (eBioScience, San Diego, CA); and Ly6G (1A8), CD45R (RA3-6B2), CD19 (1D3), CD21/35 (7G6), CD317 (927), CD44 (Pgp1), CD8 (TIB105), and CD4 (GK1.5) (purified and fluorophore-conjugated in-house). Peanut agglutinin (PNA) was from Vector Laboratories (Burlingame, CA). All antibodies and PNA were titered prior to experimental use. Cells were fixed with 1xPBS 1% paraformaldehyde. Flow cytometry data was collected on a BD LSRII or BD Fortessa and analyzed in FlowJo (Tree Star, Ashland, OR).
6
Multicolor Flow Cytometry for Conjunctival Immune Cells
7
Multiparametric Flow Cytometry Analysis
8
Multiparameter Flow Cytometry Analysis
9
Multiparametric Flow Cytometry Analysis
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Tetramer-enriched samples were stained for surface markers for 30 minutes on ice using antibodies to: CD4 (GK1.5, BD), CD8 (53–6.7, BD), CD90.1 (HIS51), CD90.2 (53–2.1, 30-H12), CD3e (145-2C11, BD), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7, BD), CD45.1 (A20, Biolegend), CD45.2 (104), B220 (RA3–6B2), and/or PD1 (J43). Cellular viability was confirmed using GhostDye Red 780 (Tonbo Biosciences). For transcription factor expression analysis, stained cells were fixed and permeablized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Cells were stained overnight at 4°C with antibodies to T-bet (4B10, Biolegend), Foxp3 (FJK-16s), RORγt (Q31-378, BD), and Bcl-6 (K112-91, BD). For thymic dendritic cell and thymic epithelial cell analyses cells, following enrichment cells were stained with antibodies to CD11c (N418), CD19 (6D5, Biolegend), I-A/I-E (M5/114.15.2, Biolegend), CD8 (53–6.7, BD), CD90.2 (53–2.1, 30-H12), CD11b (M1/70), and CD45.2 (104, BD). Antibodies were purchased from eBioscience unless otherwise indicated. Cells were analyzed by flow cytometry on a Fortessa (BD). Data were analyzed using FlowJo software (TreeStar).
10
Evaluating Dendritic Cell Maturation and Antigen Uptake
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Cells were stimulated with LPS (1 µg/ml) derived from Escherichia coli (0111:B4; Sigma-Aldrich, St. Louis, MO) at 37°C for 24 h. Cells (5×105) were collected, washed and incubated with Fc Block at 4°C for 5 min. They were labeled with APC-conjugated anti-CD40 (HM40-3, eBioscience), FITC-anti-CD80 (16-10.A1, eBioscience), PE-Cy7-anti-CD86 (GL1, BD), APC-MHC-class I (H2Kb) (AF6-88.5.5.3, eBioscience) and PE-Cy7-MHC class II (IA, IE) (M5/114.15.2, Biolegend, San Diego, CA), together with APC-anti-eFlour780-anti-CD11c (N418, eBioscience) or PE-anti-CD11c (HL3, BD) at 4°C for 30 min. In flow cytometry, live CD11c+ PI-negative cells were gated to evaluate maturation. To evaluate antigen uptake, cells were incubated with 10 µg/ml FITC-dextran (Sigma Aldrich) at 4°C or 37°C for 15 min. Cells were then labeled with APC-eFlour780-anti-CD11c. The capacity of CD11c+ cells in dextran uptake was evaluated.
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