Approximately 1500-2500 murine ILC1 were seeded with ~100 mechanically disrupted SIO crypts per well, resuspended in 30μl ice-cold Matrigel, pipetted onto pre-heated tissue culture plates (Nunclon) and incubated at 37°C for 15-20min prior to addition of pre-warmed basal media supplemented with 50mM B2ME (R&D), 20ng/ml rhIL-2 (Sigma), 20ng/ml rmIL-7 (R&D), and 1ng/ml IL-15 (R&D), with media changes every 2-4 days.
Rhil 2
Manufactured by Merck Group
Sourced in United States, Germany
RhIL-2 is a recombinant human interleukin-2 product manufactured by Merck Group. It is a cytokine that plays a critical role in the activation and proliferation of T cells, natural killer cells, and other immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Rmil 7, by R&D Systems (6 mentions)
Il 15, by R&D Systems (5 mentions)
Pre heated tissue culture plates, by Thermo Fisher Scientific (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fcil 7, by Merck Group (2 mentions)
Inositol, by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Merck Group (1 mentions)
Imdm medium, by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
α mem, by Merck Group (1 mentions)
Hek293t, by National Collection of Authenticated Cell Cultures (1 mentions)
Folic acid, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Nk 92, by American Type Culture Collection (1 mentions)
Dmi1 microscope, by Leica camera (1 mentions)
Clone okt3, by Thermo Fisher Scientific (1 mentions)
21 protocols using rhil 2
1
Culturing Murine ILC1s with Crypts
2
Cell Culture and Maintenance Protocols
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NK cell line NKG was established and maintained in our labratory as previously described [29 ] and cultured in α‐MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 15% horse serum (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL rhIL‐2 (Kingsley, Yixing, Jiangsu, China). NK cell line NK92 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in α‐MEM supplemented with 12.5% FBS, 12.5% horse serum, 0.2 mmol/L inositol (Sigma Aldrich, St. Louis, MO, USA), 0.1 mmol/L 2‐mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA), 0.02 mmol/L folic acid (Sigma Aldrich, St. Louis, MO, USA) and 100 U/mL rhIL‐2. The leukemia cell lines U‐937 and THP‐1 were obtained from ATCC and cultured in RPMI‐1640 medium (Hyclone, South Logan, UT, USA) containing 10% FBS. The erythroleukemia cell line K562 were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in IMDM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS. The human embryonic kidney cell line HEK293T were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in DMEM medium (Hyclone, South Logan, UT, USA) containing 10% FBS. All cell lines used in this study were maintained at 37°C in 5% CO2.
3
BST-2/Tetherin siRNA Electroporation in HIV-1-Infected PBMCs
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HIV-1-infected PBMCs underwent BST-2/Tetherin siRNA electroporation 2 h (protocol 1) or 3 days (protocol 2) after infection. A total of 107 PBMCs were pelleted and resuspended in 100 μL of prewarmed electroporation buffer [in-house prepared buffer solution 3P containing 5 mM KCl, 15 mM MgCl2, 90 mM NaCl, 10 mM glucose, 0.4 mM Ca(NO3)2, and 40 mM Na2HPO4/NaH2PO4 (pH 7.2) (64 (link))] premixed with BST-2/siRNA (5′-AAGCGTGAGAATCGCGGACAA ; NM_004335 ; Hs_BST2_5 FlexiTube siRNA; Qiagen, catalog number SI02777054) or scramble/siRNA (Ctrl_AllStrar_1; Qiagen, catalog number SI03650318) to a final concentration of 200 nM. Then, the cells were transferred to sterile 0.2-cm generic cuvettes (Mirus Biotech, catalog number MIR 50121) and electroporated with a Nucleofector 2b device (T-020 program; Lonza). After electroporation, the cells were immediately and gently resuspended in 1 mL of prewarmed RPMI medium supplemented with 5 U/mL rhIL-2 (Sigma-Aldrich) and 20% fetal bovine serum, transferred to cell culture flasks, and incubated at 37°C in 5% CO2 for 24 h. The day after electroporation, the cells were centrifuged and divided into two groups, one with and one without rhIL-27. The cells were maintained in culture for an additional 24 or 48 h for flow cytometry analysis or for an additional 5 days (protocol 1) or 8 days (protocol 2) to measure HIV-1 replication.
4
Culturing Murine ILC1s with Crypts
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Approximately 1500-2500 murine ILC1 were seeded with ~100 mechanically disrupted SIO crypts per well, resuspended in 30μl ice-cold Matrigel, pipetted onto pre-heated tissue culture plates (Nunclon) and incubated at 37°C for 15-20min prior to addition of pre-warmed basal media supplemented with 50mM B2ME (R&D), 20ng/ml rhIL-2 (Sigma), 20ng/ml rmIL-7 (R&D), and 1ng/ml IL-15 (R&D), with media changes every 2-4 days.
5
T Cell Derivation from PBMNCs
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PBMNC were grown in culture at 5 % CO2 at 37oC in incubator (New Burnswick Galaxy 170 R CO2 Incubator) for five days for T cell derivation. Approximately, 10x 105 cells were cultured in freshly prepared AIM-V media (12055-091, Invitrogen) with 300 IU/ml rhIL-2 (10799068001, Sigma Aldrich) and 10ng/ml plate bound anti CD3 antibody (OKT-3 clone, 16-0037-81, eBioscience)6 . The cells were observed daily under microscope (Leica DMI1 microscope, LAS v 4.6.1) and allowed to grow in the above mentioned culture condition for 5 days. On day 5, T cells were characterized by flow cytometry and then used for reprogramming.
6
Organoid-Immune Cell Co-Culture
7
Cytokine Production in CD38-/- Mice
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Splenocytes from WT and CD38-/- mice were left unstimulated or stimulated with 1 µg/mL of anti-CD3ε Ab (BD Biosciences, San Diego, CA, USA), 1 µg/mL of anti-CD28 Ab (BD Biosciences, San Diego, CA, USA), and 200U rhIL-2 (Sigma Aldrich) or Phorbol Myristate Acetate (50 ng/mL) plus Ionomycin (500 ng/mL) for 72 h. According to the manufacturer’s protocol, supernatant concentrations of IFN-γ and IL-10 were evaluated by Mouse ELISA MAXTM DELUXE Set (Biolegend, San Diego, CA, USA).
8
Chronic and Transient T-Cell Activation
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Freshly isolated T cells were plated in a 24‐multiwell plate (Corning) at the concentration of 1 × 106 cells/well in 2 ml of complete culture medium composed of RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% Penicillin–Streptomycin (Hyclone Laboratories, Inc.) and 1% L‐glutamine (Hyclone Laboratories, Inc.). To mimic a chronic stimulation, cells were cultured for 14 days in a humified CO2 incubator at 37°C in complete culture medium with Dynabeads® Human T‐Activator CD3/CD28 beads (ThermoFisher Scientific) (25 μl/well; bead‐to‐cell ratio of 1:1) and recombinant human interleukin‐2 (rhIL‐2; 25 U/ml; Sigma‐Aldrich). To mimic a transient stimulation, cells were cultured in the presence of CD3/CD28 beads and rhIL‐2 for 3 days and then rested in the presence of rhIL‐2 only for the remaining 11 days of culture. For both stimulation conditions, cells were collected at day 3, 7, 10, and 14. After collection, beads were magnetically removed using a BD IMag Cell Separation Magnet (BD Biosciences) prior to cell preparation for flow cytometry analysis, passaging, cryopreservation. For cell passaging, cells were resuspended in fresh complete medium for chronic or transient stimulation and replated at the same cell density as at day 0.
9
Isolation and Establishment of Infected Lymphocyte Cultures
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PBMCs were isolated by applying density gradient centrifugation using Ficoll-Paque PLUS (Cytiva, Marlbolough, MA, USA) following the sedimentation of red blood cells in 1.5% dextran (MW = 200,000–300,000, MP Biomedicals, LLC, Solon, OH, USA). To establish ILTs, the PBMCs were depleted of CD8+ cells using Dynabeads coated with anti-CD8 antibodies (Invitrogen, Carlsbad, CA, USA) and cultured for more than 3 months in the presence of 30 U/ml rhIL-2 (Shionogi Pharmaceuticals, Co., Osaka, Japan). The ILTs were maintained in RPMI1640 (Nacalai Tesque, Inc., Tokyo, Japan) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA)) and 30 U/ml rhIL-2. In monkeys #1936 and #2290, ILTs could not be induced, and alternative autologous STLV-1-infected cell lines were established by co-culturing autologous CD8+ cell-depleted PBMCs with MMC-treated ILT-1686 cells.
To establish herpesvirus papio-infected LCLs, PBMCs that had been depleted of CD3+ cells using Dynabeads coated with anti-mouse antibodies, after incubation with anti-CD3 antibody (BD Biosciences), were forced infected with herpesvirus papio in the supernatant of the S594 cell line, which is a herpesvirus papio-producing baboon B-cell line (kindly provided by Dr. Tetsuro Matano, National Institute of Infectious Diseases, Tokyo, Japan). The LCLs were maintained in RPMI1640 with 10% FBS and antibiotics.
To establish herpesvirus papio-infected LCLs, PBMCs that had been depleted of CD3+ cells using Dynabeads coated with anti-mouse antibodies, after incubation with anti-CD3 antibody (BD Biosciences), were forced infected with herpesvirus papio in the supernatant of the S594 cell line, which is a herpesvirus papio-producing baboon B-cell line (kindly provided by Dr. Tetsuro Matano, National Institute of Infectious Diseases, Tokyo, Japan). The LCLs were maintained in RPMI1640 with 10% FBS and antibiotics.
10
Rapid Expansion of Primary Immune Cells
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Tissues were dissected into fragments of approximately 2 mm3. Each fragment was plated individually in a single well of a 24-well plate and stimulated with 6000 IU/mL rhIL-2 (Roche, Basel, Switzerland) for 3 weeks (Pre-REP). A rapid expansion protocol (REP) [15 (link),19 (link)] was performed by stimulating PILs with PHA 1 µg/mL (Sigma, St. Loius, MI, USA), 3000 IU/mL rhIL2, and feeders. PILs culture media was RPMI supplemented with 5% penicillin-streptomycin (Gibco, Billings, MT, USA), 25 mM HEPES, 1% L-glutamine (Gibco), 1% nonessential amino acids (Gibco), 1% Na pyruvate (Gibco), 0.1% 2β-mercaptoethanol (Gibco), and 8% heat-inactivated human serum.
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