The monoclonal antibodies against mouse IL-2, CD8, and NK1.1 were obtained from hybridoma clones, S4B6-1, 53-6.72, and PK-136, respectively (American Type Culture Collection, USA). The recombinant mouse IL-2 (rIL-2) was purchased from BioLegend (USA). For the preparation of IL-2C, 1.5 μg of rIL-2 and 15 μg of anti-IL-2 were mixed in vitro, and then IL-2C was i.p. injected two times per every week at 3-day or 4-day intervals from 100 days of age until endpoint. For the isotype control, the same amount of purified rat IgG2a (BioLegend) was used. For depletion of CD8+-T cells or NK-T cells and NK cells, 100 μg of anti-CD8 or anti-NK1.1 was i.p. injected two times per every week at 3-day or 4-day intervals from 100 days of age until endpoint. Equal volume of PBS was used as the control. The efficacies of these administrations were confirmed by flow cytometry.
Ril 2
Manufactured by BioLegend
Sourced in United States
RIL-2 is a laboratory instrument used for rapid and efficient RNA extraction. It utilizes a silica-based membrane technology to isolate high-quality RNA from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse cd73 mab clone ty 23, by BioXCell (3 mentions)
Fitcconjugated anti cd45 30 f11, by BioLegend (3 mentions)
Rat igg2a isotype control clone 2a3, by BioXCell (3 mentions)
Fcr blocking reagent, by Miltenyi Biotec (3 mentions)
Ril 12, by BioLegend (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rat igg2a, by BioLegend (1 mentions)
Pk 136, by American Type Culture Collection (1 mentions)
Anti cd3 clone ucht1, by Thermo Fisher Scientific (1 mentions)
Anti cd28 clone cd28, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
α cd3 α cd28 immobilized on beads, by Miltenyi Biotec (1 mentions)
Live dead reagent, by Thermo Fisher Scientific (1 mentions)
Ril 18, by BioLegend (1 mentions)
Anti cd3 cd28 coated dynabeads, by Thermo Fisher Scientific (1 mentions)
11 protocols using ril 2
1
Modulating immune responses with IL-2C
2
Multiparametric Flow Cytometry Analysis
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The following monoclonal antibodies (mAbs) were used in cytometric studies (fluorochromes and clones in brackets): anti-CD3 (PE-Cy7, SK7), anti-CD4 (APC or PE-Cy5, RPAT4), anti-CD25 (PE, 2A3), anti-CD127 (PE-Cy7, AO19D5). Items were purchased from BD Biosciences, USA, or BioLegend, USA. Intracellular staining for Foxp3 (FITC, 206D) and IFN-γ (PerCP-Cy.5.5, B27) was performed with ready-to-use kits according to the manufacturer's suggestions (BioLegend, USA and BD Biosciences, USA, respectively). Additionally, the following reagents were used for the stimulation of cell cultures: anti-CD3 (clone UCHT1, eBioscience, USA), anti-CD28 (clone CD28.2, eBioscience, USA) antibodies, rIL-2 (BioLegend, USA), PMA (Sigma, Poland), ionomycin (Sigma, Poland), and GolgiStop (BD Biosciences, USA).
3
Splenic CD8 T Cell Activation and Proliferation
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Splenic CD8 T cells were magnetically enriched using an AutoMACS pro (Miltenyi Biotec) to >95% purity per manufacturer's instructions. The cells were fluorescein-labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) at 37° for 10', washed 3x with RPMI-1640 medium supplemented with 2-mercaptoethanol, L-glutamine, pyruvate, non-essential amino acids, and 10% fetal bovine serum (abbreviated as RP10 in the text). The cells were cultured at 2 × 105 cells/ml at a 1:1 ratio with α-CD3/α-CD28 immobilized on beads (Miltenyi), in the presence of combinations of 10 U/ml rIL-2, 10 ng/ml rIL-12 (Biolegend), and 10 ng/ml rIL-18 (Biolegend), as indicated, for up to 3 days. On each day of analysis, cells were stained for viability (LIVE/DEAD reagent, Invitrogen), as well as with surface and/or intracellular antibodies.
4
Adoptive Transfer of Tumor-Specific T Cells
5
Immunology Study in C57BL/6 Mice
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Mice were obtained from Japan SLC and maintained in specific pathogen-free conditions in our animal facility. All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee at Waseda University (#2017-A007a, 2018-A090, 2019-A041, 2020-A057, 2021-A003, A22-007, and A23-007). We used 7- to 12-wk-old male or female C57BL/6 mice. Mouse rIL-1β, rIL-2, rIL-4, rIL-6, rIL-21, rIL-23, rTGF-β1, rCXCL13, and anti-ICOS (clone C398.4A) monoclonal Abs (mAbs) were obtained from BioLegend. Mouse rIL-12 and Cellstain CFSE (5- or 6-(N-Succinimidyloxycarbonyl)fluorescein 3’,6’-diacetate) were from Fujifilm Wako Pure Chemistry. mAbs to CD28 (clone 37.51), IL-2 (clone JES6-1A12), IL-4 (clone 11B11), IL-2Rα (clone PC-61.5.3), IL-2Rβ (clone TM-β1), and IFN-γ (clone XMG1.2) were from Bio X Cell. CH-223191, 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) were from Cayman Chemical.
6
Evaluating CACT Knockdown in PBMCs
7
Ginseng Extract Enhances NK Cell Therapy
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KRG extract was used as CheongKwanJang (Korea Ginseng Corporation, Daejeon, Korea). KRG extract was made by decocting KRG roots cultivated over six years and contained ginsenoside Rg1, Rb1, and Rg3 (5 mg/g). KRG extract (50 mg/kg BW/10 mL) or water (control group) was orally administered daily after transplantation of SK-Hep1_Luc cells.
NK-92 cells (human NK cells, ATCC, CRL-2407) were maintained in alpha minimum essential medium (Gibco, Waltham, MA, USA) supplemented with 20% FBS (GW Vitek), 1% penicillin-streptomycin (GenDEPOT), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, Inc., St. Louis, MO, USA), and 100–200 U/mL rIL-2 (Biolegend, San Diego, CA, USA). NK cells (2 × 106 cells/100 μL) or phosphate-buffered saline (PBS; control group) were intraperitoneally injected twice a week beginning on day 20.
NK-92 cells (human NK cells, ATCC, CRL-2407) were maintained in alpha minimum essential medium (Gibco, Waltham, MA, USA) supplemented with 20% FBS (GW Vitek), 1% penicillin-streptomycin (GenDEPOT), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, Inc., St. Louis, MO, USA), and 100–200 U/mL rIL-2 (Biolegend, San Diego, CA, USA). NK cells (2 × 106 cells/100 μL) or phosphate-buffered saline (PBS; control group) were intraperitoneally injected twice a week beginning on day 20.
8
Characterization of Murine T Cell Subsets
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Anti-mouse CD73 mAb (clone TY/23) and Rat IgG2a isotype control (clone 2A3) were purchased from BioX-Cell (West Lebanon, NH, USA). Anti-mouse mAbs for owcytometric analysis were used as described here. FITC-conjugated anti-CD4 (GK1.5), anti-CD8α (53 - 6.7), anti-CD11b (M1/70), and PE-conjugated anti-CD39 (Duha59), anti-IFN-γ (XMG1.2), APC-conjugated anti-CD73 (TY/11.8), anti-CD3 (17A2), and FITC-conjugated anti-CD45 (30-F11), and mouse recombinant Interleukin-2 (r-IL-2) were purchased from BioLegend (San Diego, CA USA). FcR blocking reagent was obtained from Miltenyi Biotec GmBH (Bergisch Gladbach, Germany). 7AAD (7-Aminoactinomycin D) and FVS-780 were purchased from Thermo-Fisher Scienti c (Waltham, MA USA) and BD Biosciences (Franklin Lakes, NJ USA), respectively.
9
Antibody-based Immune Cell Analysis
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Anti-mouse CD73 mAb (clone TY/23) and Rat IgG2a isotype control (clone 2A3) were purchased from BioX-Cell (West Lebanon, NH, USA). Anti-mouse mAbs for owcytometric analysis were used as described here. FITC-conjugated anti-CD4 (GK1.5), anti-CD8α (53-6.7), anti-CD11b (M1/70), and PE-conjugated anti-CD39 (Duha59), anti-IFN-g (XMG1.2), APC-conjugated anti-CD73 (TY/11.8), anti-CD3 (17A2), and FITCconjugated anti-CD45 (30-F11), and mouse recombinant Interleukin-2 (r-IL-2) were purchased from BioLegend (San Diego, CA USA). FcR blocking reagent was obtained from Miltenyi Biotec GmBH (Bergisch Gladbach, Germany). 7AAD (7-Aminoactinomycin D) and FVS-780 were purchased from Thermo-Fisher Scienti c (Waltham, MA USA) and BD Biosciences (Franklin Lakes, NJ USA), respectively.
10
Antibody-based Immune Cell Analysis
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Anti-mouse CD73 mAb (clone TY/23) and Rat IgG2a isotype control (clone 2A3) were purchased from BioX-Cell (West Lebanon, NH, USA). Anti-mouse mAbs for owcytometric analysis were used as described here. FITC-conjugated anti-CD4 (GK1.5), anti-CD8α (53-6.7), anti-CD11b (M1/70), and PE-conjugated anti-CD39 (Duha59), anti-IFN-g (XMG1.2), APC-conjugated anti-CD73 (TY/11.8), anti-CD3 (17A2), and FITCconjugated anti-CD45 (30-F11), and mouse recombinant Interleukin-2 (r-IL-2) were purchased from BioLegend (San Diego, CA USA). FcR blocking reagent was obtained from Miltenyi Biotec GmBH (Bergisch Gladbach, Germany). 7AAD (7-Aminoactinomycin D) and FVS-780 were purchased from Thermo-Fisher Scienti c (Waltham, MA USA) and BD Biosciences (Franklin Lakes, NJ USA), respectively.
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