Every week beginning from week three or four, 100 µL of the medium was substituted with the same volume of fresh complete NK cell medium containing 100 unit/mL of recombinant IL-2 (Sigma-Aldrich). 104/mL of irradiated K562-mbIL21 feeder cells were added once after six weeks of clone cultivation (Figure 2 B).
Recombinant il 2
Manufactured by Merck Group
Sourced in United States
Recombinant IL-2 is a laboratory product that functions as a cytokine. It is a recombinant form of the naturally occurring human interleukin-2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Complete rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by PanEco (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque gradient, by Merck Group (1 mentions)
Pha p, by Merck Group (1 mentions)
Ifn γ, by Merck Group (1 mentions)
Tnf α, by Merck Group (1 mentions)
Anti tnf α mab11, by BD (1 mentions)
Gentlemacs system, by Miltenyi Biotec (1 mentions)
Spleen dissociation kit, by Miltenyi Biotec (1 mentions)
Mouse cd8a t cell isolation kit, by Miltenyi Biotec (1 mentions)
Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions)
Nk 92, by Merck Group (1 mentions)
8 pcpt 2 o me camp, by Merck Group (1 mentions)
Farage, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Horse serum, by Merck Group (1 mentions)
Nk 92, by American Type Culture Collection (1 mentions)
Nk 92, by Thermo Fisher Scientific (1 mentions)
15 protocols using recombinant il 2
1
Expansion of NK Cell Clones
2
Expansion of Primary Human NK Cells
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The complete NK cell medium consisted of 80% DMEM medium (PanEco, Moscow, RF), 20% xx-vivo medium (Lonza, Basel, Switzerland), or AIM-V medium (Gibco-Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 units/mL of recombinant IL-2 (Sigma-Aldrich, St. Louis, MO, USA). This medium was mixed with 104 of irradiated K562-mbIL21 feeder cells per mL and filled into 60 central wells of a 96-well plate (200 µL). The marginal wells were filled with RPMI-1640 medium to minimize evaporation from the central wells during cultivation of sorted NK cells.
3
HIV-1 Infection Assay in Activated PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors’ buffy coats (Red Cross of Luxembourg, Luxembourg, Luxembourg) using Ficoll-Hypaque gradient as indicated previously (Sigma-Aldrich, Liège, Belgium). PBMCs were stimulated using 10 μg/mL phytohemagglutinin (PHA-P, Sigma Aldrich) for 48 h and recombinant IL-2 (10 U/mL, Roche, Sigma-Aldrich, Liège, Belgium) for another 24 h. Stimulated PBMCs were infected by the HIV-1 reference strains IIIB/ADA-M or primary clinical isolates expanded in culture from anonymized left-over samples (Centre Hospitalier de Luxembourg, Luxembourg, Luxembourg) in the presence or absence of drugs replaced every other day during 7 days. P24 production was measured in supernatants by ELISA (Perkin Elmer, Brussels, Belgium). Efavirenz (EFV) and azidothymidine (AZT) were obtained from Sigma_Aldrich. Enfuvirtide (T20) was purchased from Eurogentec (Seraing, Belgium).
4
Lymphocyte Activation by Tag7 and Cytokines
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Lymphocyte activation was induced by Tag7 and IL-2 or IFNγ added to a final concentration of 10–9 M. Recombinant Tag7 (PGLYRP1) was prepared as described [4 (link)]. Recombinant IL-2, TNFα and IFNγ were from Sigma (St. Louis, MO, USA).
5
Multimodal Immune Modulation Assay
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The novel polyclonal Ig preparation, trimodulin (∼23% IgM, ∼21% IgA and
∼56% IgG), was kindly provided by Biotest AG (Dreieich, Germany).
Purified Escherichia coli LPS 0111:B4,
Staphylococcus aureus-derived lipoteichoic acid
(LTA), ConA and recombinant IL-2 were obtained from Sigma-Aldrich (St.
Louis, MO).
Fluorescently labelled Abs for flow cytometry were purchased from the
following manufacturers: anti-human leukocyte Ag–DR isotype (HLA-DR
G46-6-PE) and anti-TNF-α (mAB11) from BD Biosciences (San Jose, CA);
anti-TLR2 (CD282-PE) and anti-TLR4 (HTA125-PE) from eBioscience (San
Diego, CA); anti-cluster of differentiation 16 (CD16; 3G8-PE),
anti-CD64 (22-PC5), anti-CD11b (Bear1-PC5), anti-CD11c (BU15-PC5),
anti-CD3 (UCHT1-PC7), anti-CD4 (13B8.2-PC5) and anti-CD8
(SFCI21Thy2D3-ECD) from Beckman Coulter (Fullerton, CA); and anti-CD14
(My4; FITC, PC5 or PC7 conjugated) from Beckman Coulter (Indianapolis,
IN).
∼56% IgG), was kindly provided by Biotest AG (Dreieich, Germany).
Purified Escherichia coli LPS 0111:B4,
Staphylococcus aureus-derived lipoteichoic acid
(LTA), ConA and recombinant IL-2 were obtained from Sigma-Aldrich (St.
Louis, MO).
Fluorescently labelled Abs for flow cytometry were purchased from the
following manufacturers: anti-human leukocyte Ag–DR isotype (HLA-DR
G46-6-PE) and anti-TNF-α (mAB11) from BD Biosciences (San Jose, CA);
anti-TLR2 (CD282-PE) and anti-TLR4 (HTA125-PE) from eBioscience (San
Diego, CA); anti-cluster of differentiation 16 (CD16; 3G8-PE),
anti-CD64 (22-PC5), anti-CD11b (Bear1-PC5), anti-CD11c (BU15-PC5),
anti-CD3 (UCHT1-PC7), anti-CD4 (13B8.2-PC5) and anti-CD8
(SFCI21Thy2D3-ECD) from Beckman Coulter (Fullerton, CA); and anti-CD14
(My4; FITC, PC5 or PC7 conjugated) from Beckman Coulter (Indianapolis,
IN).
6
Isolation and Activation of Mouse T Cell Subsets
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All procedures were performed according to the protocol provided by Miltenyi Biotec. The spleen was dissociated using a Spleen Dissociation Kit (Miltenyi Biotech) and homogenized using the Miltenyi GentleMACS system. Splenocytes were further processed using the mouse CD8a+ T Cell Isolation Kit and the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotech). Isolated cells were cultured at a density of 1.5 × 106/mL in complete RPMI media with recombinant IL-2 (Sigma-Aldrich, St. Louis, Missouri) at 2500 U/mL. Cells were seeded at a density of 1.5 × 106/mL in a 24-well flat-bottom plate. Isolated cells were activated overnight with Gibco Dynabeads Mouse T-Activator CD3/CD28 for T Cell Expansion and Activation (Thermo Fisher Scientific, Inc.). Cytotoxic T cells were activated at a T cell:bead ratio of 1:1 and Treg cells at a Treg:bead ratio of 1:2.
7
Antiviral drug efficacy and cytotoxicity assay
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Human PMBCs were incubated with 100 U recombinant IL-2 (Sigma-Aldrich) and 5 µg/mL phytohemagglutinin-P (PHA-P) (Difco) for 5 days at 37°C. MT-4 cells (1×106) or treated PBMCs (1.2×108) were infected with HIV-1 clone NL-4.3 or ZM247Fv-1 (MOI of 0.001) by spinoculation (1200 g at room temperature for 2 h). Uninfected cells were used as negative control and were also subjected to the spinoculation process. After spinoculation, supernatants were discarded and cells were resuspended in 10 mL of R10. Cells from each condition were then added to wells (96-well plate) containing serial dilutions of each ISD. For MT-4 cells, each dilution was done in sextuplicates and, for PBMCs, in triplicates. Cells were also incubated with different concentrations of ZDV as positive control. After 5 days in culture, Cell Titer Blue was added to each well and cells were incubated for additional 24 h. The median effective concentration for each drug is defined as EC50. In parallel with the infectivity assay, the same cell types were treated with different concentrations of drugs to determine their cytotoxicity, following the protocol described above. Drug concentration that results in a 50% decrease in viable cells is defined as the CC50 for each compound. EC50 was calculated according to Spearman-Karber and therapeutic index was defined by the ratio between CC50 and EC50[55] (link).
8
Modulation of NHL Cell Lines
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NHL cell lines Toledo, NK-92, RL, and Farage were all purchased from American Type Culture Collection (VA, USA). Toledo, Farage, and RL cells were cultured in RPMI-1640 medium containing 10% FBS (Gibco, CA, USA); NK-92 cells were cultured in Alpha Minimum Essential Medium with ribonucleosides and deoxyribonucleosides free while with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 100–200 U/mL recombinant IL-2, 12.5% horse serum (Sigma-Aldrich Co., St Louis, MO, USA), and 12.5% FBS.
Toledo or NK-92 cells were incubated with different concentrations of forskolin (0, 10, 20, 40, 80, or 160 μM; cAMP analog 8-pCPT-2′-OMe-cAMP; Sigma-Aldrich Co) for the indicated times, and 20 μM of SP600125 (Selleck Chemicals, Shanghai, China), an inhibitor of c-Jun N-terminal kinase (JNK) for 24 hours.
Toledo or NK-92 cells were incubated with different concentrations of forskolin (0, 10, 20, 40, 80, or 160 μM; cAMP analog 8-pCPT-2′-OMe-cAMP; Sigma-Aldrich Co) for the indicated times, and 20 μM of SP600125 (Selleck Chemicals, Shanghai, China), an inhibitor of c-Jun N-terminal kinase (JNK) for 24 hours.
9
Culturing NKTCL cell lines
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The NKTCL cell lines KHYG-1 (EBV negative) and NKYS (EBV positive) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml recombinant IL2 (Sigma‒Aldrich, USA), 100 U/mL penicillin and 100 g/mL streptomycin (Gibco, USA). KAI3 (EBV positive) cells were kept in RPMI 1640 medium with 20% FBS and 100 U/ml rIL2. Cell lines were maintained at 37°C in a humidified incubator containing 5% CO2. All three cell lines were obtained from Dr Wing C. Chan (City of Hope Medical Center).
10
Sorting and Culturing of Distinct NK Cell Subsets
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