PBMCs from HLA-A*02:01 patients were used to generate DCs, which were subsequently pulsed with 20 μg/ml of MART-1 26-35 A27L (ELAGIGILTV) peptide (MBL, Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) at 37 °С for 1 h. After washing with AIM-V medium, DCs were treated with mitomycin C (25 μg/ml; Kyowa Hakko Kogyo Co, Ltd, Tokyo, Japan) at 37 °C for 1 h. After two washes, the prepared DCs were used as stimulator cells; non-adherent PBMCs collected after seeding into plastic tissue-culture plates were used as responder cells. Stimulator (1 × 106) and responder cells were co-cultured at a ratio of 1:10 in AIM-V medium supplemented with IL-2 (2.5 U/ml; Imunace, Shionogi, Pharmaceutical, Osaka, Japan), IL-7 (5 ng/ml; R&D Systems) and IL-15 (10 ng/ml; PeproTech, Rocky Hill, NJ, USA) for 3−5 days. AIM-V media supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific K.K., Yokohama, Japan) was added depending on cell expansion. After a 2–3-day incubation, cells were harvested and stained with FITC-conjugated anti-CD8 (Beckman Coulter, Inc., Brea, CA, USA) and APC-conjugated anti-CD3 (eBioscience) mAbs and T-select HLA-A*02:01 MART-1 tetramer-ELAGIGILTV-PE (MBL).
Apc conjugated anti cd3
Manufactured by Thermo Fisher Scientific
Sourced in United States
APC-conjugated anti-CD3 is a monoclonal antibody that binds to the CD3 complex, a component of the T-cell receptor. The antibody is conjugated with allophycocyanin (APC), a fluorescent dye, for detection and analysis purposes.
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Lab products found in correlation
Fitc conjugated anti cd4, by Thermo Fisher Scientific (2 mentions)
Pe conjugated anti ifn γ, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated anti cd8, by Beckman Coulter (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by R&D Systems (1 mentions)
Pe conjugated anti mhc 2, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti pd l1, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti pd 1, by Thermo Fisher Scientific (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Rabbit anti ym1, by STEMCELL (1 mentions)
Apc conjugated anti f4 80, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd206, by BioLegend (1 mentions)
Pe conjugated anti il 5, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Percp cy5 conjugated anti cd45, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti il 4, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Pe conjugated anti cd8, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti nk1, by Thermo Fisher Scientific (1 mentions)
7 protocols using apc conjugated anti cd3
1
Generating MART-1-specific CD8+ T cells
2
Flow Cytometry Analysis of Immune Cells
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Cell suspensions were prepared from spleen, lymph nodes, VAT, or liver of ob/ob mice as previously described (Chang et al., 2015 (link); Xiang et al., 2016 (link)). DC single-cell suspensions were obtained from bone marrow following standard procedures. Cells were stained with antibodies against the following cell surface antigens: fluorescein isothiocyanate (FITC)-conjugated anti-CD86, allophycocyanin (APC)-conjugated anti-CD11c, phycoerythrin (PE)-conjugated anti-MHC-II and PE-conjugated anti-PD-L1 (eBioscience, USA); APC-conjugated anti-Foxp3, FITC-conjugated anti-CD4, PE-conjugated anti-CD25 (eBioscience, USA); APC-conjugated anti-CD3, PE-conjugated anti-PD-1, and PE-conjugated anti-TCRβ (eBioscience, USA). Samples were detected on a BD LSRII flow cytometer or BD C6 (BD Pharmingen), and data were analyzed with Flowjo software, version 10.1.
3
Multiparametric Immune Cell Profiling
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Single-cell suspensions were preincubated with FcγR-specific blocking mAb (2.4G2) and washed before staining. Cells were stained with the following antibodies Percp-cy5-conjugated anti-CD45 (eBioscience), APC-conjugated anti-F4/80 (eBioscience), PE-cy7-conjugated conjugated anti-CD11c (eBioscience), FITC–conjugated anti-CD206 (BioLegend), APC-conjugated anti-CD3 (eBioscience), FITC–conjugated anti-CD4 (eBioscience); For intracellular staining, cells were permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and incubated with PE–conjugated anti-IL-4, PE–conjugated anti-IL-5, PE–conjugated anti-IFN-γ (eBioscience), rabbit anti-Ym-1 (Stem Cell Technologies) and PE–conjugated anti-rabbit IgG (eBioscience). Cells were analyzed on a LSRII (BD Biosciences) with FlowJo ver.10 software (TreeStar).
4
Flow Cytometry Analysis of Immune Cell Subsets
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LP cells from colon or splenic immune cells were sorted using a FACS flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo_V10 (Treestar, Ashlan, OR). The gating strategy has been shown in supplementary Figure 8. For macrophage and neutrophil analyses, cells were incubated with the following antibodies: FITC-conjugated anti-CD11b (11-0112-82, eBioscience, San Diego, CA), APC-conjugated anti-CD11c (17-0114-81, eBioscience), PE-cyanine7-conjugated anti-F4/80 (25-4801-82, eBioscience), PE-conjugated anti-CD206 (12-2061-80, eBioscience), and PE-conjugated anti-Ly6G (12-5931-81, eBioscience); for T cell analyses, cells were incubated with the following antibodies: APC-conjugated anti-CD3 (17-0032-82, eBioscience), FITC-conjugated anti-CD4 (11-0042-81, eBioscience), and PE-conjugated anti-CD8 (12-0081-81, eBioscience); for B cell and NK cell analyses, cells were incubated with the following antibodies: FITC-conjugated anti-CD19 (11-0193-81, eBioscience), and PE-conjugated anti-NK1.1 (12-5941-81, eBioscience).
5
MDSC Suppression of T Cell Proliferation
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Freshly isolated tumor cells from TC-1 tumors were stained with the following fluorophore-conjugated antibodies: PeCy7-conjugated anti-CD11b, FITC-conjugated anti-Gr1 and APC-conjugated anti-CD3 (eBioscience, catalog nr. 25-0112-82, 11-5931-82, 17-0031-82). Cells were washed and sorted on a FACS MoFloAstrios (Beckman Coulter) based on forward and sideward scatter. Cells were positively sorted into CD11b+Gr1+ MDSCs and negatively sorted into CD3+ T cells. As determined by flow cytometric re-analysis, the purity of the sorted MDSCs was >92%.
Spleens cells isolated from donor mice immunized twice with 5 × 106 SFVeE6,7 particles were cultured in 96-well round bottom plates for 7 d. On day 0 of culture, flow-sorted MDSCs, isolated from TC-1 tumors, were added at different ratios to these cultures. On day 4 of culture, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, catalog nr. C34554) for 10 min at 37oC. On day 5 of co-culture, recombinant IL-2 was added to the co-culture at a concentration of 5U/mL. As negative controls, splenocytes were cultured without stimulant. As positive controls, splenocytes were stimulated and cultured in the absence of MDSCs. At the end of the co-culture period, cells were collected and used for analysis.
Spleens cells isolated from donor mice immunized twice with 5 × 106 SFVeE6,7 particles were cultured in 96-well round bottom plates for 7 d. On day 0 of culture, flow-sorted MDSCs, isolated from TC-1 tumors, were added at different ratios to these cultures. On day 4 of culture, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, catalog nr. C34554) for 10 min at 37oC. On day 5 of co-culture, recombinant IL-2 was added to the co-culture at a concentration of 5U/mL. As negative controls, splenocytes were cultured without stimulant. As positive controls, splenocytes were stimulated and cultured in the absence of MDSCs. At the end of the co-culture period, cells were collected and used for analysis.
6
Quantification of Th1 and Th17 Cells
7
Immunophenotyping and Proliferation Assay
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Fas ligand (FasL) antibodies (SC-33716, Santa Cruz Biotechnology, USA) and anti-β-actin antibody (A1978, Sigma-Aldrich, USA) were used. Allophycocyanin (APC)-conjugated anti-IFN-γ, phycoerythrin (PE)-conjugated anti-IL-17, APC-conjugated anti-CD3, APC-conjugated anti-CD25, and peridinin chlorophyll protein complex (Percp)-conjugated anti-CD4 antibodies were purchased from eBioscience. Anti-CD45, CD73, CD90, CD105, and CD146 conjugated with PE and anti-CD34 and STRO1 conjugated with FTIC were purchased from BD Biosciences (Franklin Lakes, USA). BrdU solution and BrdU imaging kit were purchased from Invitrogen (Carlsbad, CA, USA).
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