Canine whole blood was obtained from healthy client-owned canines treated at Texas A&M University College of Veterinary Medicine on an institutionally approved protocol. Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Greiner Bio-One, Monroe, NC) gradient centrifugation. Canine PBMCs were stimulated with γ-irradiated (100 Gy) K562-APCs (2:1 T-cell to K562-APC ratio), 5 ug/ml PHA (Sigma-Aldrich), and 30 ng/ml recombinant human IL-21 (rhIL-21; eBioscience) in 24-well tissue culture plates and maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum and 1% GlutaMax and cultured at 38.5°C in 5% CO2. Expanded canine T cells were re-stimulated on Day 7 of culture as previously described with the addition of 100 units recombinant human IL2 (rhIL-2; Proleukin; Chiron, Emeryville, CA) and at a T cell to K562-APC ratio of 1:5 in G-Rex culture devices. Cells were fed every 3–4 days with fresh media and cytokines.
Rhil2 proleukin
Manufactured by Novartis
Sourced in Switzerland
RhIL2 (Proleukin) is a recombinant human interleukin-2 product used in laboratory research. It functions as a growth factor that stimulates the proliferation and activation of T cells and natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Lymphoprep, by Greiner (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Thermo Fisher Scientific (1 mentions)
Nk cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Ficoll hypaque gradient, by Merck Group (1 mentions)
Glutamine, by Euroclone (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Bristol-Myers Squibb (1 mentions)
Human ab serum hs, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Pulmozyme, by Roche (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
5 protocols using rhil2 proleukin
1
Canine PBMC Expansion for Immunotherapy
2
Culture and Isolation of Neuroblastoma Cells and NK Cells
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The MYC-N amplified neuroblastoma (NB) cell line HTLA-230 was provided by Dr. E. Bogenmann (Children’s Hospital Los Angeles, CA) (Corrias et al., 1996 (link)) and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FCS (Biochrom, Berlin, Germany), 50 mg/ml streptomycin, 50 mg/ml penicillin (Sigma-Aldrich), and 2 mm glutamine (Euroclone). The cells were cultured in a humidified environment (95% air/5% CO2) at 37°C and were used to generate 3D tumor models.
Peripheral blood mononuclear cells (PBMCs) were obtained from blood of volunteer healthy donors by Ficoll-Hypaque gradients (Sigma Aldrich). NK cells were purified by using the NK-cell isolation kit II (Miltenyi Biotec) and were cultured on irradiated PBMCs in RPMI-1640 supplemented with 10% heat-inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin (Sigma-Aldrich), 2 mm glutamine (Euroclone), 600 IU/ml rhIL-2 (Proleukin; Chiron, Emeryville, CA) and 0.5% v/v phytohemagglutinin (Gibco, Paisley, United Kingdom). After 10 passages, NK cells were checked for purity (>95%) analyzing classical NK cell markers (Castriconi et al., 2007b (link)).
Peripheral blood mononuclear cells (PBMCs) were obtained from blood of volunteer healthy donors by Ficoll-Hypaque gradients (Sigma Aldrich). NK cells were purified by using the NK-cell isolation kit II (Miltenyi Biotec) and were cultured on irradiated PBMCs in RPMI-1640 supplemented with 10% heat-inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin (Sigma-Aldrich), 2 mm glutamine (Euroclone), 600 IU/ml rhIL-2 (Proleukin; Chiron, Emeryville, CA) and 0.5% v/v phytohemagglutinin (Gibco, Paisley, United Kingdom). After 10 passages, NK cells were checked for purity (>95%) analyzing classical NK cell markers (Castriconi et al., 2007b (link)).
3
Expansion of Allogeneic T Cells
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Human AB serum (HS) was purchased from Sigma- Aldrich (Brøndby, Denmark); RPMI-1640 with GlutaMAX and AIM-V medium were obtained from Invitrogen (Nærum, Denmark). RhIL2 (Proleukin) was from Novartis (Basel, Switzerland), and OKT3 (anti-CD3) antibody was from Cilag AG (Schaffausen, Switzerland). Pulmozyme was purchased from Roche (Basel, Switzerland). Allogeneic peripheral blood mononuclear cells (PBMCs) (or feeder cells) were obtained from buffy coats from healthy donors. Solu-Cortef (hydrocortisone sodium succinate) was obtained from the local hospital pharmacy. Fetal bovine serum (FBS) was from Gibco (Nærum, Denmark). Complete medium (CM) consisted of RPMI-1640 with GlutaMAX, 25 mM HEPES pH 7.2, 100 U/ml penicillin, 100 μg/ml streptomycin and Fungizone® (Bristol-Myers Squibb, New York, NY, USA) 1,25 lg/ml supplemented with 10% HS and 6000 IU/ml of rhIL2. Rapid expansion medium (RM) consisted of AIM-V medium and Fungizone® 1,25 lg/ml supplemented with 6000 IU/ml rhIL2.
4
NOD Mice IL-2 Dose Response
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Five- to fourteen-week-old female and male NOD mice were treated with daily intraperitoneal injections of 250,000; 500,000 or 1,000,000 IU of rhIL-2 (Proleukin, Novartis) for 30 consecutive days.
5
Expansion of Natural Killer Cells
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Expansion of NK cells was performed as described previously (29 (link)). Briefly, PBMCs, prepared from peripheral blood using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden), were co-cultured with gamma-irradiated (100 Gy) KL-1 and LCL feeder cell lines in RPMI 1640 medium supplemented with 10% FBS and recombinant human IL-2 (500 U/ml; rhIL-2, Proleukin; Novartis, Basel, Switzerland). Medium was changed every 3 days up to 6 days, and every 4 days up to 18 days, thereafter. Fresh IL-2 was added when medium was changed during the culture. On day 6, expanded NK cells were transferred to T25 or T75 flasks at a concentration of 0.25 × 106 cells/ml. The absolute number of NK cells was calculated by multiplying the total number of viable cells by the percentage of CD56+CD3− cells, measured by flow cytometry. Fold change was determined by dividing the number of viable NK cells present at the designated day of culture by the number of viable NK cells at the beginning of culture.
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